Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides

ABSTRACT

A method for the stepwise creation of phosphodiester bonds between desired nucleosides resulting in the synthesis of polynucleotides having a predetermined nucleotide sequence by preparing an initiation substrate containing a free and unmodified 3&#39;-hydroxyl group; attaching a mononucleotide selected according to the order of the predetermined nucleotide sequence to the 3&#39;-hydroxyl of the initiating substrate in a solution containing a catalytic amount of an enzyme capable of catalyzing the 5&#39; to 3&#39; phosphodiester linkage of the 5&#39;-phosphate of the mononucleotide to the 3&#39;-hydroxyl of the initiating substrate, wherein the mononucleotide contains a protected 3&#39;-hydroxyl group, whereby the protected mononucleotide is covalently linked to the initiating substrate and further additions are hindered by the 3&#39;-hydroxyl protecting group. Methods in which a mononucleotide immobilized on a solid support is added to a free polynucleotide chain are also disclosed.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/300,484 filed Sep. 2, 1994.

TECHNICAL FIELD

This invention relates to the synthesis of oligonucleotides and othernucleic acid polymers using template independent enzymes.

BACKGROUND OF THE INVENTION

Oligonucleotides are presently synthesized in vitro using organicsynthesis methods. These methods include the phophoramidite methoddescribed in Adams et al., J. Amer. Chem. Soc., 105:661 (1983) andFroehler et al., Tetrahedron Lett., 24:3171 (1983) and thephosphotriester method described in German Offenlegungsshrift 264432.Other organic synthesis methods include those described by Froehler etal., U.S. Pat. No. 5,264,566 in which H-phosphonates are used to produceoligonucleotides.

The phosphoramidite method of phosphodiester bond formation andoligonucleotide synthesis represents the current state of the artemployed by most laboratories for the coupling of desired nucleotideswithout the use of a template. In this method, the coupling reaction isinitiated by a nucleoside attached to a solid support. The 5'-hydroxylgroup of the immobilized nucleoside is free for coupling with the secondnucleoside of the chain to be assembled. Since the growingoligonucleotide chain projects a 5'-hydroxyl available for reaction witha mononucleotide, the direction of synthesis if referred to as 3' to 5'.

Each successive mononucleotide to be added to the growingoligonucleotide chain contains a 3'-phosphoramidate moiety which reactswith the 5'-hydroxyl group of the support-bound nucleotide to form a 5'to 3' internucleotide phosphodiester bond. The 5'-hydroxyl group of theincoming mononucleotide is protected, usually by a trityl group, inorder to prevent the uncontrolled polymerization of the nucleosides.After each incoming nucleoside is added, the protected 5'-hydroxyl groupis deprotected, so that it is available for reaction with the nextincoming nucleoside having a 3'-phosphoramidite group and a protected5'-hydroxyl. This is followed by deprotection and addition of the nextincoming nucleotide, and so forth.

Between each nucleoside addition step, unreacted chains which fail toparticipate in phosphodiester bond formation with the desired nucleosideare chemically "capped" to prevent their further elongation. Thisusually involves chemical acetylation.

This method and the other currently used organic methods while widelyaccepted require large amounts of costly monomers that require complexorganic synthesis schemes to produce. In addition, these methods arecomplex in that the phosphoramidite method requires an oxidation stepafter each condensation reaction. The phosphotriester method requiresthat the subpopulation of oligonucleotides that have not had a monomeradded in a particular cycle be capped in a separate reaction to preventfurther chain elongation of these oligonucleotides.

Other drawbacks of virtually all chemical methods of phosphodiester bondformation, is that the reaction must be performed in organic solventsand in the absence of water. Many of these organic solvents are toxic orotherwise hazardous. Another drawback to chemical synthesis is that itis at best 98 percent efficient at each cycle. In other words, followingeach nucleotide addition, at least 2 percent of the growingoligonucleotide chains are capped, resulting in a yield loss. The totalyield loss for the nucleotide chain being synthesized thus increaseswith each nucleotide added to the sequence.

For example, assuming a yield of 98 percent per nucleotide addition, thesynthesis of a polynucleotide consisting of 70 mononucleotides wouldexperience a yield loss of nearly 75 percent. Furthermore, the objectnucleotide chain would require isolation from a reaction mixture ofpolynucleotides, nearly 75 percent of which consist of cappedoligonucleotides ranging between 1 and 69 nucleotides in length.

This inherent inefficiency in chemical synthesis of oligonucleotidesultimately limits the length of oligonucleotide that can be efficientlyproduced to oligonucleotides having 50 nucleic acid residues or less.

A need exists for a method which improves the efficiency ofphosphodiester bond formation and which could ultimately be capable ofproducing shorter chain oligonucleotides in higher yields and longerchain polynucleotides in acceptable yields. In addition, a need existsfor a polynucleotide synthesis system which is compatible withpre-existing polynucleotides, such as vector DNAs, so that desiredpolynucleotide sequences can readily be added on to the pre-existingsequences. Chemical coupling by the phosphoramidite method is notcompatible with "add-on" synthesis to pre-existing polynucleotides.Enzyme catalyzed phosphodiester bond formation, however, can beperformed in an aqueous environment utilizing either single or doublestranded oligo- or polynucleotides to initiate the reaction. Thesereaction conditions also greatly minimize the use of toxic and hazardousmaterials.

The 3' to 5' direction of synthesis inherent to the phosphoramiditemethod of phosphodiester bond formation cannot be enzyme catalyzed. Allknown enzymes capable of catalyzing the formation of phosphodiesterbonds do so in the 5' to 3' direction since the growing polynucleotidestrand always projects a 3'-hydroxyl available for attachment of thenext nucleoside.

There are many enzymes capable of catalyzing the formation ofphosphodiester bonds. One class of such enzymes, the polymerases, arelargely template dependent in that they add a complementary nucleotideto the 3' hydroxyl of the growing strand of a double strandedpolynucleotide. However, some polymerases are template independent andprimarily catalyze the formation of single stranded nucleotide polymers.Another class of enzyme, the ligases, are template independent and forma phosphodiester bond between two polynucleotides or between apolynucleotide and a mononucleotide.

Addition of single nucleotides to DNA fragments, catalyzed bydeoxynucleotidyl terminal transferase (TdTase), has previously beendescribed by Deng and Wu, Meth. Enzymol., 100:96-116, 1983. Thesereaction conditions did not involve transient protection of the3'-hydroxyl nor were they intended to be used for the sequentialcreation of phosphodiester bonds to synthesize a predeterminednucleotide sequence. The presence of unprotected 3'-hydroxyls resultedin a highly heterogeneous population of reaction products.

Similarly, prior attempts to catalyze synthesis of very short pieces ofRNA or DNA using protected nucleotide monophosphates or diphosphatesresulted in unacceptably low levels of the desired phosphodiester bondformation or required excessive amounts of enzyme to achieve acceptableefficiencies. These problems were largely due to unavoidableheterogeneity of the mononucleotide building blocks or to the very highturnover number of the enzyme, necessitating extremely long incubationtimes (see, for example, Hinton and Gumport, Nucleic Acids Res.7:453-464, 1979; Kaufman et al., Eur. J. Biochem., 24: 4-11, 1971).These experiments were limited to 5'-monophosphates and diphosphates. Noattempts have been made to catalyze controlled DNA synthesis using5'-triphosphates protected at the 3' position.

Enzyme catalyzed creation of a single phosphodiester bond between thefree 3'-hydroxyl group of an oligonucleotide chain and the 5'-phosphateof a mononucleotide requires protection of the 3'-hydroxyl of themononucleotide in order to prevent multiple phosphodiester bondformations and hence repeated mononucleotide additions. Protection ofthe 3'-hydroxyl of the mononucleotide ideally involves a transientblocking group which can readily be removed in order to allow subsequentreactions. Flugel et al., Biochem. Biophys. Acta. 308:35-40, 1973,report that nucleoside triphosphates with blocked 3'-hydroxyl groupscannot be prepared directly. This lack of 3' blocked triphosphatesnecessitated previous processes to utilize lower energy and thus moreinefficient 3' blocked monophosphates and diposphates. Synthetictechniques to create 3' block triphosphates would be highly desirable,because this could enable stepwise enzyme catalyzed phosphodiester bondformation leading to polynucleotide synthesis.

These prior attempts at synthesizing oligonucleotides using a templateindependent polymerase were extremely inefficient resulting in theproduction of very short oligonucleotides. The inefficiency of thesemethods made these methods useless for practical synthesis ofoligonucleotides.

The present invention allows the creation of phosphodiester bondsbetween nucleotides using a template independent polymerase to createoligonucleotides having a predetermined sequence. This enzyme catalysiscan vastly improve the efficiency of phosphodiester bond formationbetween desired nucleotides compared to current techniques of chemicalcoupling and can be carried out in the presence of other biologicalmolecules such as pre-existing sequences of single or double strandedDNA as well as other types of enzymes. In addition, the very highspecificity inherent to enzyme catalysis allows only coupling of a5'-phosphate to a 3'-hydroxyl. The coupling of two mononucleosides, aswell as various other side reactions inherent to chemical couplingtechniques, simply do not occur.

A further advantage of the present invention is realized by using 3'blocked triphosphates having high energy phosphate bonds which an enzymecan utilize to drive the reaction to greater completion level than whenother monophosphates and diphosphates are used. In addition,triphosphates are less strongly hydrated than the diphosphate, whichalso tends to drive catalytic hydrolysis of the triphosphate tocompletion.

Clearly, the availability of a homogeneous population of protectedmononucleotide triphosphates and enzymes capable of efficiently joiningprotected nucleotides to initiating substrates will enable the creationof a highly uniform population of synthetic polynucleotides resultingfrom stepwise phosphodiester bond formation.

SUMMARY OF THE INVENTION

A number of methods have been discovered by which the 3'-hydroxyl groupof a deoxynucleotide triphosphate can be effectively protected anddeprotected and wherein the protected nucleotide is utilized by atemplate independent polymerase to create a phosphodiester bondpermitting the synthesis of oligonucleotides or polynucleotides having adesired predetermined sequence.

Therefore, in accordance with the present invention, a method isprovided for the synthesis of a polynucleotide of a predeterminedsequence of which method includes the steps of:

A. providing an initiating substrate comprising a nucleoside having anunprotected 3'-hydroxyl group; and

B. reacting under enzymatic conditions in the presence of a catalyticamount of an enzyme the 3'-hydroxyl group of the initiating substratewith a nucleoside 5'-triphosphate having a removable blocking moietyprotecting the 3' position of the nucleoside 5'-triphosphate andselected according to the order of the predetermined sequence, so thatenzyme catalyzes the formation of a 5' to 3' phosphodiester linkagebetween the unprotected 3'-hydroxyl group of the initiating substrateand the 5'-phosphate of the nucleoside 5'-triphosphate to produce thepolynucleotide.

In other embodiments of the present invention, the method furthercomprises the step:

C. removing the blocking moiety protecting the 3' position of saidnucleotide 5'-triphosphate to produce an initiating substrate having anunprotected 3'-hydroxyl group.

In other embodiments, steps (b) and (c) are repeated at least once toadd additional nucleotides to the initiating substrate by alternativelyadding a nucleoside 5'-triphosphate with a removable blocking moiety atits 3' position, deblocking the 3' position of the terminal nucleosideand then adding another nucleoside 5'-triphosphate with a removableblocking group at its 3' position. Repetition of steps (b) and (c) canalso be carried out to produce an oligonucleotide or polynucleotidehaving a predetermined sequence.

The present invention contemplates initiating substrates that aredeoxynucleosides, nucleotides, single or double strandedoligonucleotides, single or double stranded polynucleotides,oligonucleotides attached to nonnucleoside molecules and the like.

The present invention contemplates embodiments in which the substrate isimmobilized on a solid support. Preferred solid supports includecellulose, Sepharose, controlled-pore glass, silica, Fractosil,polystyrene, styrene divinyl benzene, agarose, and crosslinked agaroseand the like.

The present invention contemplates the use of template independentpolynucleotide polymerases such as terminal deoxynucleotidyl transferasefrom any number of sources including eukaryotes and protharyotes.

The methods of the present invention utilize removable blocking moietiesthat block the 3' position of nucleoside 5'-triphosphates used in themethods. Preferred removable blocking moieties can be removed in under10 minutes to produce a hydroxyl group at the 3' position of the 3'nucleoside. Removable blocking groups contemplated includecarbonitriles, phosphates, carbonates, carbamates, esters, ethers,borates, nitrates, sugars, phosphoramidates, phenylsulfenates, sulfatesand sulfones.

The methods of the present invention contemplate removing the removableblocking moiety using a deblocking solution that preferably containsdivalent cations such as Co++ and a biological buffer such as comprisesa buffer selected from the group consisting of dimethylarsinic acid,tris hydroxymethyl! amino methane, and 3- m-morpholine! propanosulphonicacid. Other embodiments of the present invention utilize an enzymepresent in the deblocking solution to remove the removable blockingmoiety.

The present invention also contemplates methods in which the nucleoside5'-triphosphate having the removable blocking moiety at its 3' positionis immobilized in a solid support and reacted with free initiatingsubstrates. The solid support is linked to the nucleoside5'-triphosphate at the 3'-hydroxyl group, thereby acting as a removableblocking moiety at the 3' position. Attachment of the nucleoside to thesupport is transient, thereby enabling the release of the newlysynthesized product from the support and regeneration of the free andunmodified 3'-hydroxyl to allow the next nucleotide addition to occur.

Thus, in some embodiments of the present invention the deblockingsolution would remove the removable blocking moiety at the position ofthe nucleoside and thus release the growing polynucleotide from thesolid support.

The present invention also includes polynucleotides having apredetermined sequence provided according to the methods of thisinvention. Applications for using polynucleotides and oligonucleotidesof the present invention in molecular cloning and/or expression ofgenes, peptides or proteins.

Also contemplated by the present invention are compositions of mattercomprising a catalytic amount of a template independent enzyme and anucleoside 5'-triphosphate having a removable blocking moiety protectingthe 3' position of said nucleoside 5'-triphosphate. Additionalcompositions of matter further comprising an initiating substrate arealso contemplated.

BRIEF DESCRIPTION OF THE INVENTION

A. Definitions

DNA: Deoxyribonucleic acid.

RNA: Ribonucleic acid.

Nucleotide: A subunit of a nucleic acid comprising a phosphate group, a5-carbon sugar and nitrogen containing base. In RNA, the 5-carbon sugaris ribose. In DNA, it is a 2-deoxyribose. The term also includes analogsof such subunits.

Nucleoside: Includes a nucleosidyl unit and is used interchangeablytherewith, and refers to a subunit of a nucleic acid which comprises a5-carbon sugar and a nitrogen containing base. The term includes notonly those nucleosidyl units having A, G, C, T and U as their bases, butalso analogs and modified forms of the naturally-occurring bases, suchas pseudoisocytosine and pseudouracil and other modified bases (such as8-substituted purines). In RNA, the 5-carbon sugar is ribose; in DNA, itis 2'-deoxyribose. The term nucleoside also includes other analogs ofsuch subunits, including those which have modified sugars such as2'-O-alkyl ribose.

Polynucleotide: A nucleotide multimer generally about 50 nucleotides ormore in length. These are usually of biological origin or are obtainedby enzymatic means.

Phosphodiester: The group ##STR1## wherein phosphodiester groups may beused as internucleosidyl phosphorus group linkages (or links) to connectnucleosidyl units.

Hydrocarbyl: An organic radical composed of carbon and hydrogen whichmay be aliphatic (including alkyl, alkenyl, and alkynyl groups andgroups which have a mixture of saturated and unsaturated bonds),alicyclic (carbocyclic), aryl (aromatic) or combination thereof; and mayrefer to straight-chained, branched-chain, or cyclic structures or toradicals having a combination thereof, as well as to radicalssubstituted with halogen atom(s) or heteroatoms, such as nitrogen,oxygen, and sulfur and their functional groups (such as amino, alkoxy,aryloxy, lactone groups and the like), which are commonly found inorganic compounds and radicals.

Non-nucleoside monomeric unit: A monomeric unit wherein the base, thesugar and/or the phosphorus backbone or other internuclosidyl linkage ofa nucleoside has been replaced by other chemical moieties.

Polypeptide and Peptide: A linear series of amino acid residuesconnected on to the other by peptide bonds between the alpha-amino andcarboxyl groups of adjacent residues.

Protein: A linear series of greater than about 50 amino acid residuesconnected one to the other as in a polypeptide.

Gene: A segment of DNA coding for an RNA transcript that is itself astructural RNA, such as ribosomal RNA or codes for a polypeptide. Thesegment of DNA is also equipped with a suitable promoter, terminationsequence and optionally other regulatory DNA sequences.

Structural Gene: A gene coding for a structural RNA and being equippedwith a suitable promoter, termination sequence and optionally otherregulatory DNA sequences.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a gene and to which RNApolymerase specifically binds and initiates RNA synthesis(transcription) of that gene.

Oligonucleotide: A chain of nucleosides which are linked byinternucleoside linkages which is generally from about 2 to about 50nucleosides in length. They may be chemically synthesized fromnucleoside monomers or produced by enzymatic means. The termoligonucleotide refers to a chain of nucleosides which haveinternucleosidyl linkages linking the nucleoside monomer and, thus,includes oligonucleotide containing nucleoside analogs, oligonucleotidehaving internucleosidyl linkages such that one or more of thephosphorous group linkages between monomeric units has been replaced bya non-phosphorous linkage such as a formacetal linkage, a thioformacetallinkage, a sulfamate linkage, or a carbamate linkage. It also includesnucleoside/non-nucleoside polymers wherein both the sugar and thephosphorous moiety have been replaced or modified such as mopholino baseanalogs, or polyamide base analogs. It also includesnucleoside/non-nucleoside polymers wherein the vase, the sugar, and thephosphate backbone of a nucleoside are either replaced by anon-nucleoside moiety or wherein a non-nucleoside moiety is insertedinto the nucleoside/non-nucleoside polymer. Thus an oligonucleotide maybe partially or entirely phophonothioates, phosphorothioatephosphorodithioate phosphoramidate or neutral phosphate ester such asphosphotriesters oligonucleotide analogs.

Removable Blocking Moiety: A removable blocking moiety is a moiety whichis attached to the oxygen at the 3' position of a nucleoside or theequivalent position in a nucleoside analog. The removable blockingmoiety prevents reaction of the 3' oxygen when present and is removableunder deblocking conditions so that the 3' oxygen can then participatein a chemical reaction.

A. Methods

Generally, the present invention provides methods for synthesizingoligonucleotides and polynucleotides having a predetermined sequenceusing a template independent polymerase and nucleoside having the 3'position blocked with a removable blocking moiety so that singlenucleosides are added to the growing oligonucleotide. Single nucleosidesare added to the growing chain by removing the blocking moiety at the 3'position of the terminal nucleoside of the growing oligonucleotide sothat the next blocked nucleoside can be added to the oligonucleotide.This process is then repeated until the oligonucleotide having thepredetermined sequence is produced.

Thus, in accordance with this embodiment of the present invention, amethod comprises the steps of:

(a) providing an initiating substrate comprising a nucleoside having anunprotected 3'-hydroxyl group; and

(b) reacting under enzymatic conditions in the presence of a catalyticamount of an enzyme said 3'-hydroxyl group of said initiating substratewith a nucleoside 5'-triphosphate having a removable blocking moietyprotecting the 3' position of said nucleoside 5'-triphosphate andselected according to the order of said predetermined sequence, wherebysaid enzyme catalyzes the formation of a 5' to 3' phophodiester linkagebetween said unprotected 3'-hydroxyl group of said initiating substrateand the 5'-phosphate of said nucleoside 5'-triphosphate to produce saidpolynucleotide.

In preferred embodiments, the methods of the present invention furthercomprises the step of:

(c) removing the blocking moiety protecting the 3' position of saidnucleoside 5'-triphosphate to produce an initiating substrate having anunprotected 3'-hydroxyl group.

This additional step regenerates a reactive atom at the 3' position ofthe terminal nucleoside so that this atom can be used to form a bondwith the next nucleoside and thus extend the length of theoligonucleotide by one nucleoside.

The methods of the present invention also include methods in which theabove steps (b) and (c) are repeated at least once to produce anoligonucleotide. This process can be repeated many times to produceoligonucleotides of selected length. This process can also be repeatedmany times such that each particular nucleoside added to theoligonucleotide having a preselected sequence.

1. Initiating Substrates

An initiating substrate of the present invention is prepared containinga nucleoside with a free and unmodified 3'-hydroxyl group. As is wellunderstood by those of ordinary skill in the art, nucleotide derivativesof the nucleosides adenosine, cytidine, guanosine, uridine and thymidinecan be assembled to form oligonucleotides and polynucleotides. Accordingto the method of the present invention, the initiating substrate maycontain a single nucleoside having a free and unmodified 3'-hydroxylgroup, or a preassembled oligo- or polynucleotide may be provided as aninitiating substrate, so long as the oligo- or polynucleotide has a freeand unmodified 3'-hydroxyl group.

One skilled in the art will understand that an initiating substratecould be provided in a form in which a nucleoside has a removableblocking moiety at its 3' position which is subsequently removed using adeblocking process so that the initiating substrate now has the freeunprotected 3' hydroxyl group useful in the present invention.

The initiating substrates of the present invention include the terminiof polynucleotides frequently generated and used in various cloning andmolecular biology techniques. Examples of these initiating substratesinclude the termini of DNA or RNA vectors, single-stranded ordouble-stranded fragments, single-stranded or double-stranded RNAfragments and RNA or DNA oligonucleotides.

In the preferred embodiments, initiating substrates will consist whollyor in part of an oligo- or polynucleotide. The initiating substrate canbe any arrangement of nucleosides which enables the enzyme to create aphosphodiester bond between the 3'-hydroxyl of a nucleoside and the5'-phosphate of a mononucleotide. Initiating substrates may be basedwholly or in part on ribonucleic acids (RNA) or deoxyribonucleic acids(DNA) and may be single stranded or multi-stranded. In addition,initiating substrates can include other types of naturally occurring orsynthetic molecules (non-nucleosides) which may enable or enhance theability of the enzyme to create a phosphodiester bond or which mayfacilitate the manipulation of reaction components and by-products. Anexample of this would be a linker molecule (commonly used linkersconsist of C, O, N, and H e.g. Affi-Gel™ 10: R--OCH₂ CONH(CH₂)₂NHCO(CH₂)₂ COON(CH₂)₂ which would serve to provide a convenient methodfor attaching an initiating substrate to a solid support.

The sequential creation of phosphodiester bonds and hence the additionof nucleotides to the initiating substrate may be performed entirely insolution, or the initiating substrate may be attached to an insolublematrix. Attachment to an insoluble matrix will permit the rapidseparation of the substrate from unreacted reagents in order to preparethe substrate for the addition of the next nucleotide. For this reason,the substrate is preferably affixed to a solid support matrix duringeach reaction creating a phosphodiester bond.

Insoluble matrices suitable for use as solid supports include cellulose,Sepharose™, controlled-pore glass (CPG), polystyrene, silica, agarose,and the like.

Reagents, buffers and solvents suitable for use with the presentinvention are capable of flowing through the solid support matrix, bywhich means the initiating substrate is brought into contact with thesematerials. The growing nucleotide chain remains attached to the solidsupport as the various reagents, buffers and solvents sequentially flowtherethrough. The solid support matrix is preferably contained within asynthesis column, to which reagents, buffers and solvents are provided.

Attachment of the initiating substrate to the solid support can be bycovalent bonding. Numerous methods for the covalent attachment ofmolecules to insoluble matrices have been described and are wellunderstood by those of ordinary skill in the art. In the preferredembodiment an oligonucleotide chain may be linked to alkylaminederivatized polystyrene or CPG by way of a covalent phosphoramidate bondalthough numerous strategies for linking oligonucleotides to solidsupports have been described. The choice of an appropriate linkingstrategy will depend on the specific requirements of stability, chargeinteractions, solubility and the like.

Alternatively, attachment of the initiating substrate to the solidsupport can be by non-covalent interactions. Numerous methods for thetransient attachment of molecules to insoluble matrices have beendescribed and are well understood by those of ordinary skill in the art.For example, an oligonucleotide derivative containing single or multiplebiotin molecules may be attached to avidin-agarose orstreptavidin-agarose to form a non-covalent linkage between theoligonucleotide and the insoluble agarose matrix.

In general, it is envisioned that single and double stranded oligo- andpolynucleotides based on DNA or RNA may be covalently or non-covalentlybound to solid supports to form a variety of initiating substrates.Regardless of the strategy employed to attach an initiating substrate toan insoluble matrix, a nucleoside with a free and unmodified 3'-hydroxylgroup will always be available for enzyme catalyzed creation of aphosphodiester bond.

2. Template Independent Enzymes

Mononucleotides are added to the free and unmodified 3'-hydroxyl groupof the initiating substrate by reacting the substrate with the5'-phosphate of the selected mononucleotide in the presence of acatalytic amount of an enzyme capable of creating the phosphodiesterbond covalently linking the 5'-phosphate of the mononucleotide with the3'-hydroxyl of the substrate. The enzyme is preferable a templateindependent enzyme such as a template independent polynucleotidepolymerase. Template independent enzymes such as template independentpolynucleotide polymerases are capable of catalyzing the formation of aphosphodiester bond between the nucleotides without requiring thepresence of a complementary nucleotide strand for activity. Thus, thetemplate independent enzymes such as template independent polynucleotidepolymerases are able to catalyze the formation of single-strandednucleic acid polymers without requiring a complementary nucleic acidstrand to act as a template. Examples of template independentpolynucleotide polymerases include terminal deoxynucleotidyltransferases. Template independent polynucleotide polymerases can beisolated from a number of sources including calf thymus and othersources of lymphocytes. A particularly preferred polymerase is terminaldeoxynucleotidyl transferase (TdTase, EC 2.7.7.31).

Enzymes capable of being utilized with the present invention can bereadily identified by those of ordinary skill in the art, and areemployed under appropriate and well understood conditions. Exampleenzymatic conditions for deoxynucleotidyl transferase include a pH of6.8 maintained by a potassium cacodylate buffer, 8 mmol/l of MgCl₂, 1mmol of β mercaptoethambol, 0.33 mmol/l of ZnSO₄. One skilled in the artwill understand that these enzymatic conditions may vary while stillallowing the enzyme to catalyze the desired reaction.

3. Nucleosides Having Removable Blocking Moieties

In accordance with the present invention, the mononucleotide has its 3'position protected by a removable blocking moiety so that a singlephosphodiester linkage is formed between the free 3'-hydroxyl of theinitiating substrate and the 5'-phosphate group of the mononucleotide.The removable blocking moiety protecting the 3' position of themononucleotide prevents the catalytic creation of multiplephosphodiester bonds and hence multiple nucleotide additions.

The present invention contemplates a nucleoside 5'-phosphate of thepresent invention has a removable blocking moiety protecting the 3'position having the following formula: ##STR2## wherein R2 istriphosphate, diphosphate or monophosphate; and wherein R1 is ahydrocarbyl. In preferred embodiments the nucleoside 5' phosphate of theabove formula has an R2 group which is triphosphate and an R1 groupwhich is a hydrocarbyl.

Nucleotides having a removable blocking moiety protecting the 3'position suitable for use with the present invention have a structurecorresponding to Formula 1, that has a structure which is compatiblewith the utilization of the entire nucleotide for the creation of aphosphodiester bond by the enzyme. ##STR3##

B is the nucleotide base and R₂ represents the appropriate mono-, di- ortriphosphate. R₁ can be an ester linkage, COR₁ ', which forms thestructure nucleotide-3'-O--CO--R₁ '. R₁ ' can be any alkyl or aryl groupcompatible with the utilization of the molecule by the enzyme for thecreation of an internucleotide phosphodiester bond. The chemistry ofesters as protecting groups for hydroxyls is well established. Removableblocking moieties including formate, benzoyl formate, acetate,substituted acetate, propionate, isobutyrate, levulinate, crotonate,benzoate, napthoate and many other esters have been described in detail(See, Greene, T. W., Protective Groups in Organic Chemistry, John Wiley& Sons, New York, 1981). Esters in general are readily removed, usuallyin the presence of base, to regenerate the hydroxyl group and thus areuseful as removable blocking moieties.

Ester removable blocking moieties are formed by reacting the nucleotidewith the appropriate acid anhydride. Alternatively, a carboxylic acidcan be esterified with the 3'-hydroxyl of the nucleotide in the presenceof water after activation by reaction with carbonyl diimidazole (See,Schafer et al., Meth Enzymol., 126, 682-712.)

The present invention also contemplates a nucleoside 5'-phosphate havinga removable blocking moiety protecting the 3' position which is an esterand which has the following formula: ##STR4## wherein R2 istriphosphate, diphosphate or monophosphate; and wherein R1 is anyaliphatic or aromatic organic ester.

The present invention also contemplates a nucleoside 5'-phosphate havinga removable blocking moiety protecting the 3' position which is an esterand which has the following formula: ##STR5## wherein R is selected fromthe group consisting of: formate, benzoylformate, chloroacetate,dicholoroacetate, trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,phenylacetate, 3-phenylpropionate, 3-benzoylpropionate, isobutyrate,monosuccinoate, 4-oxopentanoate, pivaloate, adamanioate, crotonate,4-methoxycrotonate, (E)-2-methyl-2-butenoate, o-(dibromomethyl)benzoate,o-(methoxycarbonyl)benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoateand α-naphthoate.

The present invention also contemplates a nucleoside 5'-phosphate havinga removable blocking moiety protecting the 3' position which is an esterand which has the following formula: ##STR6## wherein R2 istriphosphate, diphosphate or monophosphate; and wherein R is selectedfrom the group consisting of: H, CH₃, CH₃ (CH₂)_(N) where N is aninteger from 1 to 12, (CH₃)_(x+1) (CH)_(x) where x is an integer from 1to 12, (CH₃)_(x+1) (CH)_(x) (CH₂)_(n) where x and n are independentintegers from 1 to 12, C_(x) (CH₃)_(3x-)(x-1) (CH2)_(n) where x and nare independent integers from 1 to 12, ##STR7## where R1, R3, R4, R5 andR6 is CH₃, H or NO₂.

In preferred embodiments, the nucleoside 5' phosphates of the presentinvention are deoxynucleoside 5' triphosphate having a removableblocking moiety protecting the 3' position which can be any of theblocking groups disclosed in this specification or equivalents of thosegroups.

An alternative type of removable blocking moiety utilizes an etherlinkage which forms the structure nucleotide--3'-O--R'. In this instanceR'₁ can be methyl, substituted meythyl, ethyl, substituted ethyl, butyl,allyl, cinnamyl, benzyl, substituted benzyl, anthryl or silyl. Thechemistry involved in using ethers as removable blocking moieties forhydroxyls is well known in the art. Numerous ethers have been describedand are useful for transiently protecting hydroxyls and similar chemicalgroups.

In other embodiments, a nucleoside 5' phosphate of the present inventionhas a removable blocking moiety protecting the 3' position which is anether and which has the following formula: ##STR8## wherein R2 istriphosphate, diphosphate or monophosphate; and wherein R1 is an etherselected from the group consisting of a substituted or unsubstituted:aliphatic group, aromatic group or silyl group.

In other embodiments, a nucleoside 5' phosphate of the present inventionhas a removable blocking moiety protecting the 3' position which is anether and which has the following formula: ##STR9## wherein R2 istriphosphate, diphosphate or monophosphate; and wherein R1 is an etherselected from the group consisting of a methyl, methoxymethyl,methylthiomethyl, benzyloxymethyl, t-butoxymethyl,2-methoxyethoxymethyl, 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl,tetrahydropyarnyl, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl,4-methoxytetrahydropranyl, 4-methoxytetrahydrothiopyranyl,4-methoxytetrahydrothiopyranyl S,S-dioxido, tetrahydrofuranyl,tetrahydrothiofuranyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl,1-(isopropoxy)ethyl, 2,2,2-trichloroethyl, 2-(phenylselenyl)ethyl,butyl, allyl, cinnamyl, p-chlorophenyl, benzyl, p-methoxybenzyl,o-nitrobenzyl, p-nitrobenzyl, p-halobenzyl, p-cyanobenzyl,3-methyl-2-picolyl N-oxido, diphenylmethyl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl,p-(p'-bromophenacyloxy)phenyldiphenylmethyl, 9-anthryl,9-(9-phenyl-10-oxo)anthryl, benzisothiazolyl S,S-dioxido,trimethylsilyl, triethylsilyl, isopropyldimethylsilyl,t-butyldimethylsilyl, (triphenylmethyl)dimethylsilyl,methyldiisopropylsilyl, methyldi-t-butylsilyl, tribenzylsilyl,tri-p-xylylsilyl, triisopropylsilyl and triphenylsilyl.

In preferred embodiments, a nucleoside 5' phosphate of the presentinvention has a removable blocking moiety protecting the 3' positionwhich is an ether and which has the following formula: ##STR10## whereinR2 is triphosphate, diphosphate or monophosphate; and wherein R1 isselected from the group consisting of bis(2-chloroethoxy)methyl ether,4-methoxytetrahydropyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethylether, tri(p-methoxyphenyl)methyl ether, di(p-methoxy)phenylmethylether, t-butyldimethylsily ether.

In more preferred embodiments, a nucleoside 5'phosphate of the presentinvention has a removable blocking moiety protecting the 3' positionwhich is an ether and which has the following formula: ##STR11## whereinR2 is triphosphate, diphosphate or monophosphate; and wherein R1 is CH₃,CH₃ (CH₂)_(N) where N is an integer from 1-10, methyl, methoxymethyl,methoxyethoxymethyl, trimethlsilyl, and triethylsilyl. In a morepreferred embodiment, the nucleoside 5'-phosphate of the presentinvention has an R1 group which is CH(OC₂ H₅)CH₃ and R2 is triphosphateand said nucleoside 5'-phosphate is a deoxynucleoside.

Additional well known removable blocking moieties useful for protectingfor hydroxyls include carbonitriles, phosphates, carbonates, carbamates,borates, nitrates, phosphoramidates, and phenylsulfenates. Most of thesechemical modifications to the nucleotide can be removed by chemicalreactions. Some modifications may also be removed by enzymatic digestionresulting in the regeneration of the 3' hydroxyl. These would includephosphates, glycosides, and certain esters.

In other embodiments, a nucleoside 5'-phosphate of the present inventionhas a removable blocking moiety protecting the 3' position having thefollowing formula: ##STR12## wherein R2 is triphosphate, diphosphate ormonophosphate; and wherein R1 is selected from the group consisting ofphosphate, phosphoramidate and phosphoramide. In preferred embodimentsthe nucleoside 5' phosphate of the above formula has an R2 group whichis triphosphate and an R1 group which is phosphate.

More preferred embodiments of the present invention contemplate anucleoside 5'-phosphate having a removable blocking moiety protectingthe 3' position which has the following formula: ##STR13## wherein R2 istriphosphate, diphosphate or monophosphate; and wherein R1 is selectedfrom the group consisting of phosphate, toluic acid ester or ethoxyethylether.

The present invention also contemplates a nucleoside 5'-triphosphatehaving a removable blocking moiety protecting the 3' position which isan ester produced by directly esterifying the nucleoside5'-triphosphate. In preferred embodiments the nucleoside 5'-triphosphatehas an ester is benzoate or acetate as the removable blocking moietyprotecting the 3' position. In the more preferred embodiments, thenucleoside 5'-triphosphate produced by direct esterification is adeoxynucleoside.

The present invention also contemplates a nucleoside 5'-triphosphatehaving a removable blocking moiety protecting the 3' position which isan ether produced by directly attaching an ether to the nucleoside5'-triphosphate.

Particularly preferred are deoxynucleoside 5'triphosphates produced bydirectly attaching an ether to the 3' position.

Attachment of the nucleotide having a removable blocking moietyprotecting the 3'-position to the free and unmodified 3'-hydroxyl of theinitiating substrate is then accomplished by reacting incubating! theaforementioned nucleotide and the substrate with an enzyme capable offorming a phosphodiester bond between the two. Specifically, this bondwould link the 5'-phosphate of the mononucleotide with the 3'-hydroxylof the initiating substrate. This reaction can be performed either freein solution or, in one embodiment of the invention, the initiatingsubstrate is immobilized on a solid support.

Particularly preferred are removable blocking moieties and deblockingreaction conditions that allow the blocking moiety to be removed inunder 10 minutes to produce a hydroxyl group at the 3' position of the3'-terminal nucleoside. Other preferred removable blocking moieties anddeblocking conditions allow the blocking moiety to be removed in lessthan 8, 7, 6, 5, 4, 3, 2, or 1 minutes,

4. Reactions

In preferred embodiments, the preferred enzyme is TdTase, and specificexamples of uses of this enzyme are set forth below. However, thepresent invention should not be considered limited to the TdTasecatalyzed synthesis of DNA and use of other enzymes capable ofcatalyzing the formation of a 5' to 3' phosphodiester linkage betweenthe 3' hydroxyl group of the substrate and the 5' phosphate of thenucleoside having the removable blocking moiety is contemplated by thepresent invention. One skilled in the art will understand that enzymereaction conditions are selected to allow the desired catalysis to occurand may be performed under appropriate conditions, and these conditionsare well known in the art.

The reacting is performed typically between 25° C. and 42° C. for anappropriate period of time, typically between about one minute and about30 minutes. Very short reaction times may be particularly useful if theremovable blocking moiety is unstable.

For TdTase catalyzed reactions, the enzymatic conditions, which mayserve as the solution in which the substrate is reacted, contains fromabout 0.20 and about 200 μM of the nucleotide having the removableblocking moiety protecting the 3'-hydroxyl, and from about 0.20 to 200μM of free and unmodified 3'-hydroxyls derived from the initiatingsubstrate. One particularly preferred buffer contains from about 10 toabout 500 mM potassium cacodylate buffer (pH between 6.5 and 7.5), andfrom about 0.01 to about 10 mM of a divalent cation (e.g. CoCl₂ orMnCl₂). Other buffer compositions and components may be suitable forparticular desired embodiment of the present invention.

For example, enzymatic conditions for deoxynucleotidyl transferaseinclude a pH of 6.8 maintained by a potassium cacodylate buffer, 8mmol/l of MgCl₂, 1 mmol·l of β mercaptoethanol, 0.33 mmol/l of ZnSO₄.One skilled in the art will understand that these enzymatic conditionsmay vary while still allowing the enzyme to catalyze the desiredreaction.

The enzyme capable of catalyzing the formation of 5' to 3'phosphodiester linkages between the 3' hydroxyl group of the initiatingsubstrate and the 5' phosphate of the nucleoside being added is presentin a catalytic amount. A catalytic amount of enzyme is typicallysufficient to catalyze the formation of phosphodiester bond betweengreater than 99% of the free 3' hydroxyls of the initiating substrateand the 5' phosphate of the nucleoside within 1 hour. Preferably, thecatalytic amount of enzyme and the enzymatic conditions are such thatgreater than 99% of the free 3' hydroxyls of the initiating substrateare reacted within 10 minutes. In other preferred embodiments, thecatalytic amount of enzyme and the enzymatic conditions are such thatgreater than 90% of the free 3' hydroxyls of the initiating substrateare reacted in less than 5 minutes, for example 4, 3, 2 or 1 minutes. Inother preferred embodiments, the catalytic amount of enzyme andenzymatic conditions are such that greater than 99% of the free 3'hydroxyls of the initiating substrate are reacted within 2 minutes.

The TdTase enzyme is present in the buffer at a level between about 1and 200 units per μL. One unit of TdTase catalyzes the transfer of 1nmol of DATP to p(dT)₆₋₁₂ in 60 minutes at 37° C. Commercially availableforms of TdTase include calf thymus TdTase, available from a variety ofsuppliers (e.g. Sigma Chemical Co., St. Louis Mo., Promega Corp,Madison, Wis., Gibco-BRL, Gaithersburg, Md.). Calf thymus TdTase mayalso be prepared by the procedures described by Modak, Biochemistry, 17,3116-20 (1978), and by Bollum, Fed. Proc. Soc. Exp. Biol. Med. 17, 193(1958).

While the substrate containing a free and unmodified 3'-hydroxyl groupand the mononucleotide having the removable blocking moiety protectingthe 3'-hydroxyl group can be reacted in the presence of the TdTase inthe buffer solution, the substrate is preferably immobilized on a solidsupport, and more preferably in a synthesis column to which the buffersolution containing the reaction components is delivered.

After the appropriate incubation time, the enzyme, unreactedmononucleotide, buffer and divalent cation are separated from theinitiating substrate. If the reaction was performed using a free andsoluble substrate, it can be separated by conventional size exclusionchromatography or similar types of separation techniques including butnot limited to ion exchange chromatography and affinity chromatography.For initiating substrates immobilized on solid supports, separation isachieved by washing the support with water or a suitable buffer.

One advantage to the present invention is that the level of unreactedhydroxyl groups on the initiating substrate after the aforementionedenzyme reaction can be exceptionally low, less than 0.1%. This minimizesthe necessity for capping unreacted hydroxyl groups. In some embodimentsof the present invention it may be desirable to cap the unreactedsubstrates before the next step in the synthesis cycle. The appropriatechemistry for accomplishing this can be derived from any of theprotection strategies described previously but must be permanentlyaffixed during all the subsequent cycles. An example of capping isacetylation by reaction of free 3'-hydroxyls with acetic anhydride andpyridine which would be applicable when acetylation (or otheresterifications) are not used as the protecting group on themononucleotide. Alternatively capping can be accomplished by reactionwith t-butyldimethylchlorosilane in acetonitrile and pyridine to form asilyl ether which would be applicable when similar ethers are not usedto protect the mononucleotide. In the preferred embodiment, thesereactions are intended primarily for modifying immobilized initiatingsubstrates in order to rapidly and efficiently provide appropriatecapping conditions.

After the appropriate incubation time, capping reagents are separatedfrom the initiating substrate. If the reaction was performed using asoluble substrate, it can be separated by conventional size exclusionchromatography or similar types of separation techniques including butnot limited to ion exchange and affinity chromatography. For initiatingsubstrates immobilized on solid supports, separation is achieved bywashing the support with water or a suitable buffer.

The removable blocking moieties protecting the 3' position on theinitiating substrate after the reaction may be removed or deblocked(deprotected) to regenerate a free and unmodified 3'-hydroxyl availablefor addition of the next nucleotide. One skilled in the art willunderstand that this may be accomplished by either chemical or enzymaticmethods. For example, ester protecting groups may be removed using anesterase when R₁ of the ester protecting group discussed above is asuitable substrate for the esterase. Otherwise, the ester linkage may becleaved by base hydrolysis, which is accomplished by contacting theprotected 3'-hydroxyl group with a suitable concentration of base for asufficient period of time. Cleavage of ester protecting groups has beenwell studied and appropriate reaction conditions can be readilyidentified that will cleave the ester but will not cleave the linkageused for capping (e.g. an ether).

The present invention incorporates the unexpected discovery that certainremovable blocking moieties, the aromatic 3'-O esters of deoxynucleotidetriphosphates, are unstable in commonly used buffers containing divalentcations. The instability is attributable to the presence of both thebuffer and the divalent cation, and does not result from the presence ofthe buffer alone or the cation alone. Buffers destabilizing the esterprotecting groups may contain dimethylarsinic acid (cacodylic acid),tris(hydroxymethyl) aminomethane, sodium acetate and sodium phosphate.Divalent cations destabilizing to ester blocking groups include cobalt,manganese and magnesium ions. The toluic acid ester of a deoxynucleotidetriphosphate is unstable in a mixture of 1 mM CoCl₂, 100 μM potassiumcacodylate, pH 6.8.

Conditions for the removal of removable blocking moieties such asethers, carbonates, nitrates, and other protecting groups are wellstudied and many are compatible with the integrity of a polynucleotidechain. Removal of blocking moieties such as phosphate protecting groups,the hydroxyl is regenerated by enzymatic digestion with a phosphatase.For removal of blocking moieties when the protecting group is a sugarmoiety, regeneration of the hydroxyl can be accomplished by enzymatichydrolysis using a glycosidase.

If the removal or deblocking reaction is performed in solution, thedeprotection reagents are simply added to the solution. If the reactionis performed with the initiating substrate immobilized on a solidsupport, then the hydroxyl group regeneration step is performed bywashing the solid support with the deprotection reagents. When synthesiscolumns are utilized to contain the solid support, the hydroxyl groupregeneration step is performed by washing the column with theappropriate agents.

After the appropriate period for removal, the initiating substrate(including both those that received an additional nucleotide and thosethat are capped) is again separated from the other reaction components.If the reaction was performed using a soluble substrate, it can beseparated by conventional size exclusion chromatography or similar typesof separation techniques including but not limited to ion exchange andaffinity chromatography. For initiating substrates immobilized on solidsupports, separation is achieved by washing the support with water or asuitable buffer.

As will be appreciated, the above described steps of enzyme catalyzedphosphodiester bond formation between a nucleotide having a removableblocking moiety at its 3' position and an initiating substrate,separation of the initiating substrate from reaction components, cappingof unreacted initiating substrate, again separating the initiatingsubstrate from reaction components, removing the removable blockingmoiety to regenerate the 3'-hydroxyl group, and again separating theinitiating substrate from reaction components are repeated as necessaryuntil the desired object polynucleotide chain is completely synthesized.

Cleavage of a newly synthesized polynucleotide strand from the solidsupport and/or from the initiating substrate can be accomplished byeither chemical or enzymatic reactions. In the case of a chemicalreaction, if the initiating substrate terminal nucleoside (containingthe free and unmodified 3'-hydroxyl group) is a deoxyguanosinemethylated at the 7 position of the base:

Support-dCCCCCCCCCCC-Me⁷ -G-object polynucleotide (SEQ. ID No. 1)reaction with 1M piperidine in water at 90° C. will cleave the chain atthis position yielding only the desired polynucleotide in solution. Thismethod can yield a polynucleotide chain containing only thepredetermined sequence and can be performed either on immobilized chains(to effect cleavage) or on solution synthesized chains to remove theinitiating substrate. Alternatively, the dG^(7me) can be positioned atany location within the initiating substrate or the objectpolynucleotide where cleavage is desired. Other examples of modifiedbase-specific cleavage of polynucleotide chains have been extensivelydescribed in the literature (See, Ambrose and Pless, Meth. Enzymol., IVol 152: 522-538.)

Enzymatic removal of the polynucleotide chain may be accomplished byreaction with a specific restriction endonuclease. For example, if theinitiating substrate oligonucleotide has the following structure:

Support-dCCCCCCCCCCCCCCCTGCA-3'-OH (SEQ ID No. 2)

and the object polynucleotide begins with a G, the resulting newlysynthesized chain can be cleaved from the support by reaction with Pst 1restriction enzyme. This method assumes there are no additional Pst 1restriction sites in the newly synthesized chain and that one hasannealed an appropriate oligonucleotide to the Pst 1 site to render itin a double stranded form for recognition by the enzyme (e.g. anannealing oligonucleotide with the following structure:3'-dGGGGGGGGGGGGGGGACGTC-5' (SEQ ID No. 3) for the example above).Depending on the desired first nucleotide of the object polynucleotide,as well as the ultimate sequence of the polynucleotide, one can choosefrom a wide variety of restriction enzymes to accomplish the cleavage ofonly the desired sequence. This method can yield a polynucleotide chaincontaining only the predetermined sequence and can be performed eitheron immobilized chains (to effect cleavage) or on solution synthesizedchains to remove the initiating substrate. Alternatively, appropriaterestriction endonuclease recognition sequences can be positioned at anylocation within the initiating substrate or the object polynucleotidewhere cleavage is desired.

The combined initiating substrate and object polynucleotide can becleaved from the solid support by chemical methods. How the cleavage isperformed will depend upon the nature of the initiating substrate andhow it was attached to the solid support. Covalent labile bonds, such asfor example a trityl group, can be cleaved by washing the support withan appropriate protic acid. Numerous other cleavage strategies have beendescribed. In the case of a non-covalent attachment, as for exampleavidin-biotin binding, release of the combined substrate and objectpolynucleotide will occur upon incubation with 8M guanidine-HCl, pH 1.5.

If the entire synthesis was performed using a soluble initiatingsubstrate, the initiating substrate containing the object polynucleotidecan be separated from the various capped oligo- and polynucleotides byconventional chromatographic techniques, such as polyacrylamide gelelectrophoresis. Similarly, if the initiating substrate is cleaved fromthe object polynucleotide by chemical or enzymatic means (e.g. byreaction with piperidine or by restriction endonucleases digestion asdescribed above) conventional chromatography can be used to purify theobject polynucleotide.

If the synthesis was performed using an initiating substrate immobilizedto a solid support, cleavage from the solid support can be accomplishedby either chemical or enzymatic means to retrieve either the combinedinitiating substrate and object polynucleotide or the objectpolynucleotide alone. In each instance, the object polynucleotide willbe contaminated with capped oligo- and polynucleotides which can beseparated from the object polynucleotide by polyacrylamide gelelectrophoresis.

An alternative strategy for the synthesis and recovery of the objectpolynucleotide involves immobilization of the nucleotide. In thisinstance, the nucleotide is protected at the 3'-hydroxyl by a linkerwhich is attached to a solid support. The linker attachment to thenucleotide can be by an ester or by any of the aforementioned protectinggroup strategies. Solid supports containing various functional groups(e.g. amines, amides, biotin, avidin, and the like) are generallyavailable and can be adapted to the particular requirements of thenucleotide linker. For example, a nucleotide linker containing a biotinmolecule can be bound to agarose using an avidin functional groupattached to the agarose.

Using an immobilized nucleotide, the TdTase reaction would join a freeinitiating substrate, in solution, to the immobilized nucleotide,thereby immobilizing only those initiating substrates which haveparticipated in the enzyme reaction. Initiating substrates which had notparticipated in the TdTase reaction would be easily removed by rinsingthe solid support with an appropriate buffer. Regeneration of the 3'hydroxyl on the initiating substrate is accomplished by the sametechniques as described previously.

Subsequent to the regeneration and cleavage step, the initiatingsubstrate is rinsed away from the solid support and separated from theregeneration/cleavage solution containing free nucleotides byconventional techniques such as size exclusion chromatography, ionexchange or affinity chromatography. The next immobilized nucleotide,contained on a new population of solid support particles, is then mixedwith the initiating substrate and the appropriate buffers in order torepeat the TdTase coupling reaction.

By immobilizing the nucleotide rather than the initiating substrate, acapping reaction is obviated since the object polynucleotide isseparated from unreacted initiating substrate at every cycle. Similarly,if the cleavage reaction fails to release all of the objectpolynucleotide chains, those polynucleotides which continue to beattached to the solid support are removed prior to the subsequent TdTasereaction.

It is envisioned that various newly synthesized polynucleotide chainswill subsequently be joined together by a polymerase/ligase type ofreaction in order to form longer polynucleotide sequences that are in adouble stranded form. For example, newly synthesized polynucleotides Aand B may have the structures depicted below:

A: 3' - p(dN) - dCCCCCCCCC -5' (SEQ ID No. 4)

B: 3' - p(dN) - dGGGGGGGGG -5' (SEQ ID No. 5)

where p(dN) is the predetermined object polynucleotide sequence uniqueto either the A or B polynucleotide. In the presence of the Klenowfragment of DNA polymerase I, and T4 DNA ligase, as well as theappropriate buffers and nucleotides, a double stranded polynucleotidewill be formed in which the two object polynucleotides have been"stitched" together to form the longer double stranded polynucleotide C:##STR14##

This reaction can be performed when one of the polynucleotides is stillattached to a solid support or when both polynucleotides have beenreleased into solution by the techniques described previously.

B. Polynucleotides

The present invention contemplates oligonucleotides and polynucleotidesproduced using the methods of this invention. These polynucleotidespreferably have a predetermined nucleotide sequence that was produced byselecting the order in which the individual nucleotides were added tothe initiating substrate so when synthesis is completed a polynucleotidehaving a preselected sequence is produced. Alternatively, randomcombination of polynucleotides could also be produced by introducing allfour blocked nucleotides during an individual coupling reaction orduring numerous individual coupling reactions.

In preferred embodiments, a polynucleotide produced according to themethods of this invention is greater than five nucleotides in length.Polynucleotides produced according to the methods of the presentinvention may contain large numbers of nucleotides, for example 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200 and greater than 200 nucleotides. The length of apolynucleotide produced using the methods of the present invention maybe of a length that is intermediate between the aforementionednucleotide lengths, such as 5, 15, 16, 25, 26 or any other numeralintermediate between the specific lengths. The length of thepolynucleotide produced according to the present invention is limitedonly by the efficiency of the processes of the present invention.

Polynucleotides produced by the methods of the present invention cancontain nucleotide sequences that have a variety of biologic andmolecular biologic uses. One skilled in the art will understand the usesfor long polynucleotides having a predetermined sequence. For example,many manipulations commonly performed in modern molecular biology couldbe greatly simplified through the availability of inexpensive, longpolynucleotides having a predetermined sequence.

Examples of molecular biology procedures and manipulations that would besimplified using the polynucleotides produced according to the methodsof the present invention include cloning and expression of variousnucleic acids both in vitro and in vivo. For examples of techniques andmanipulations that are simplified using polynucleotides produced usingthe methods of the present invention see, Methods in Enzymology, Vol.152 edited by Berger and Kimmel; Maniatis et al., Molecular Cloning aLaboratory Manual, Cold Spring Harbor Press, 1990; Current Protocols inMolecular Biology, edited by Ausubel et al., John Haley and Sons, NewYork, 1987.

For example, polynucleotides of the present invention could be used tointroduce restriction sites into a nucleic acid, to introduce variousnucleotide sequences having biological activity such as promoters, andto adjust reading frames. The number of possible applications usingoligonucleotide and polynucleotide produced according to the presentinvention is large as one skilled in the art will understand.Oligonucleotides and polynucleotides produced according to the presentinvention are especially useful when the application requires longoligonucleotides and polynucleotides.

C. Compositions of Matter

The present invention also contemplates compositions of mattercomprising a catalytic amount of a template independent enzyme and anucleoside 5'-triphosphate having a removable blocking moiety protectingthe 3' position of the nucleoside 5' triphosphate and other preferredcompositions further comprise an initiating substrate of the presentinvention.

The present invention contemplates compositions having an amount oftemplate independent enzyme capable of catalyzing the formation of a 5'to 3' phosphodiester linkage between 99 percent of the unprotected 3'hydroxyl groups present on an initiating substrate of the presentinvention and a nucleoside 5'-triphosphate having a removable blockinggroup protecting its 3' position within 10 minutes. Other compositionsare contemplated that contain an amount of enzyme capable of performingthe same reaction to the same extent within 2 minutes.

The compositions contemplated by the present invention includescompositions in which the template independent polynucleotide enzymepresent is a template independent polynucleotide polymerase. Examples ofpreferred template independent polynucleotide polymerases include TdTaseand enzymes with similar activities.

The composition of the present invention includes a nucleoside having aremovable blocking moiety protecting the 3' position of the nucleoside.Particularly preferred are nucleoside 5'-triphosphates having aremovable blocking moiety protecting the 3' position of the nucleoside.The various useful removable blocking moieties are described herein.

In preferred compositions, the nucleoside having the removable blockingmoiety protecting the 3' position is present at a concentration of 1nanomolar to 100 mmolar. In other preferred compositions, the nucleosideis present at a concentration of 1 micromolar to 1 millimolar. In otherpreferred embodiments, the nucleoside 5'-triphosphate having theremovable blocking moiety protecting its 3' position is present at aconcentration of 10 times the Km of the enzyme present in thecomposition.

D. Automated Processes and Apparatus

The present invention contemplates the incorporation of the methoddescribed herein in an automated process in an apparatus and in devices.For example, the various buffer and reagent solutions of the inventiveprocess can be provided to synthesis columns containing initiatingsubstrates affixed to solid support matrices by the use of flexibletubing attached to peristaltic pumps or similar devices controlled by amicroprocessor programmed to meter the exact quantities of the materialsin the correct sequence.

Regardless of the equipment employed, it can be appreciated that themethod of the present invention can create single phosphodiester bondsbetween desired nucleosides with very high efficiency and canpotentially be used to produce long chain polynucleotides in highyields.

One example of such an automated process is depicted by a porous frit 13in a glass or plastic vessel 15 shown in FIG. 3. The insoluble matrix 11consists of a solid support such as cellulose, SEPHAROSE™ or CPG towhich a nucleotide, nucleoside or polynucleotide is covalently linked atthe 5'-position of the terminal nucleotide or to which an oligo- orpoly-nucleotide or nucleoside having a terminal nucleoside with a free3'-hydroxyl group is covalently attached via the 5'-hydroxyl group. Thematrix II may itself be a covalent component of the porous frit 13 or itmay be a separate entity.

The various solutions involved in the synthesis cycle are stored instock containers 21, 23, 25, 27, 28 and 29. Solutions are introducedinto the vessel 15 through tubes 41, 43, 47, 48, 49 and 50 attached topumps 31, 33, 35, 37, 38, 39 and 40. The composition of the stocksolutions would depend on the stability of the various components of themixture. A simplified automated process would combine many of thevarious reagents as follows:

The stock containers 21, 23, 25 and 27 contain buffer solutions 51, 53,55 and 57, respectively, having a concentration between about 10 andabout 500 mM of sodium cacodylate (pH 7.0 at 25° C.), between about 0.1and about 1.0 mM of dithiothreitol. Each buffer solution also containsbetween about 0.10 and about 200 units per μL of an enzyme (e.g. TdTase)suitable for phosphodiester bond formation. Buffer solution 51 in stockcontainer 21 also contains between about 0.20 and about 200 μM ofdeoxyadenosine 5'-triphosphate having a blocked 3'-hydroxyl group.Buffer solutions 53, 55 and 57 in stock containers 23, 25 and 27,respectively, contain equivalent concentrations of deoxycytosine5'-triphosphate, deoxyguanosine 5'-triphosphate and thymidine5'-triphosphate respectively, each of which also has blocked 3'-hydroxylgroups. Buffer solution 58 in stock container 28 contains an appropriatereagent for deblocking the blocked 3'-hydroxyl groups of the fournucleosides as described previously. Stock solution 59 in stockcontainer 29 contains a suitable neutralization buffer at pH 7.0, suchas 0.1M sodium cacodylate. Stock solution 30 in container 60 contains asuitable enzymatic solution or chemical reagent for releasing the finalproduct from the solid support as described previously.

The various stock solutions are drawn into the tubing, which each feedonto the matrix. Recycling of buffer solutions 51, 53, 55 and 57 fromthe vessel 15 to their respective stock containers 21, 23, 25 and 27 canbe accomplished by way of the tubing 61, 63, 65 or 67. Allocation offluid to the appropriate tubing can be accomplished by a distributor,71, which directs the fluid from the vessel 15. Distributor devices,such as multiport stopcocks and fraction collectors are familiar to oneof ordinary skill in the art. Movement of the liquid through tubingwhich is downstream from the distributor (e.g. 61, 63, 65, 67, 69, 73)can be accomplished by additional pumping as needed (e.g. pump 83, 85,87, 89, 91, 93). At least one microprocessor controls the peristalticpumps and distributor so as to provide for the sequential addition andrecycling of the nucleotides to form a nucleotide chain having apredetermined nucleotide sequence. In the preferred processes, theinitiating substrate linked to the matrix 11 is first exposed to one ofthe solutions 51, 53, 55, or 57 for a sufficient time to enableattachment of the nucleotide to the initiating substrate. This solutionis then recycled into the appropriate container (21, 23, 25, or 27).

The amount of TdTase and 5'-nucleoside triphosphate contained in thebuffer solutions 51, 53, 55 and 57 is sufficient for the synthesis of apredetermined quantity of an object nucleotide chain. For example, forthe automated synthesis of 1 nmol of a nucleotide chain consisting of1,000 bases (about 330,000 MW and about 330 μg), each buffer solutionwill contain an excess of each 5'-nucleoside triphosphate (about 500nmol) and an excess of TdTase (about 100 to about 1,000 units). Only asmall fraction of the buffer solution containing the TdTase and the5'-nucleoside triphosphate is used for each cycle of nucleotideaddition. Matrix 11 is next exposed to solution 58 for a sufficientperiod of time to remove blocking groups from the growing oligo- orpolynucleotide chain. This solution is not recycled but is distributedto tube 69 by the distributor 71, utilizing pump 91.

Matrix 11 is then briefly exposed to solution 59 in order to wash outthe deblocking reagent. The next enzyme/nucleotide solution, either 51,53, 55 or 57, is then added to matrix 11 to continue the cycle.

Finally, after the desire oligo- or polynucleotide is synthesized,cleavage of the object polynucleotide from the solid support occurs bythe controlled addition of solution 60 which can be a restrictionendonuclease solution or a solution to effect the chemical cleavage fromthe solid support (e.g., 1M piperidine) as described above. Themicroprocessor directs the distributor 71 and pump 93 to move the finalproduct through tube 73 to be recovered for final workup.

As an example of the control of the various reactions by themicroprocessor, the synthesis of the oligonucleotide ACGT onto aninitiating substrate would involve the sequence of commands shown below.The duration of and between each command is sufficient to allow anyparticular reaction or fluid movement to proceed adequately.

    ______________________________________                                        Microprocessor Command                                                                            Intended Result                                           ______________________________________                                        1.    Pump 31 on.       Solution 51 added to                                                          vessel 15                                             2.    Pump 31 off.      Nucleotide addition                                                           reaction proceeds                                     3.    Distributor 71 on,                                                                              Recycle reaction fluid                                      pump 83 on.       fluid via tube 61                                     4.    Distributor 71 off,                                                                             Solution 58 added to                                        pump 83 off.      vessel 15; initiate                                                           deblocking reaction                                   5.    Pump 38 off.      Deblocking reaction                                                           proceeds                                              6.    Distributor 71 on,                                                                              Discard deblocking fluid                                    pump 91 on.       via tube 69                                           7.    Pump 39 on.       Neutralize/wash reaction                                                      chamber                                               8.    Distributor 71 off,                                                                             Solution 53 added to                                        pump 69 off, pump vessel 15                                                   33 on.                                                                  ______________________________________                                    

This cycle is repeated for the other nucleotides until the desiredsequence is synthesized. When collection of the final product isdesired, the microprocessor gives the following commands after step 7above.

    ______________________________________                                        1.     Pump 40 on.   Solution 60 added to vessel                                                   15                                                       2.     Pump 40 off.  Cleavage reaction of the                                                      initiating substrate proceeds                            3.     Distributor 71 on,                                                                          Collection of synthesized DNA                                   pump 93 on    via tube 73                                              ______________________________________                                    

The alternative strategy envisions the use of immobilized nucleotidetriphosphates in order to separate the object nucleotide fromnon-reacting substrate polynucleotides at every cycle. The automatedprocess using immobilized nucleotide is considerably different from theprocess involving an immobilized substrate polynucleotide. After thecoupling reaction of the triphosphate and the polynucleotide, the eluatecontains unreacted polynucleotides, reaction buffer, and TdTase enzyme.The object polynucleotide is attached to the solid support. In order torecycle the enzyme back to its reservoir, the contaminatingpolynucleotide is first removed by passing the solution through a columncontaining hydroxyl apatite, for example, or a similar polynucleotideadsorption medium through which the enzyme will pass. This column willhave sufficient capacity to adsorb all of the anticipated contaminatingpolynucleotides produced by every cycle.

After the deblocking step, the object polynucleotide is now contained ina solution of nucleotide triphosphates (with 3'-hydroxyls), anddeblocking buffer (e.g., NaoH or phosphatase). These two contaminantscan be removed by size exclusion chromatography (e.g., SEPHAROSE™ CL-6B)or by any of a number of commonly used techniques for separating smallmolecules from oligo- and polynucleotides. An example of this isadsorption of the object polynucleotide by annealing to oligodA-cellulose column (3'-5') which would simply require the initiatingsubstrate to contain oligo dT. A-T annealing is the preferred embodimentin the automated process since elution of the object polynucleotide canbe accomplished by incubation with H₂ O. The annealing of the objectnucleotide simply requires the neutralization of the cleavage reactionby addition of a sufficient quantity of HCl or by the inclusion of anappropriate amount of NaCl (.sup.˜ 0.1-0.5M) in the deblocking buffers.

An automated process incorporating the immobilized nucleotidetriphosphate alternative method of the present invention is depicted inFIG. 4. The process utilizes a nucleotide triphosphate immobilized to asolid support by, but not limited to, techniques describe previously,and compromising stock solutions 151, 153, 155, 157 in stock containers121, 123, 125, 127. The stock solutions contain a tethered nucleotide,appropriate buffers and sufficient enzyme to effect the synthesis of thedesired amount of predetermined sequence. The immobilization materialhas fluid dynamic properties allowing it to be moved through the varioustubes as required. Substances which have these characteristics (e.g.gels and viscous suspensions) are familiar to one of ordinary skill inthe art. The reaction vessel, 115, contains a reaction chamber, 111, anda stopcock, 113. Stopcock 113 has three positions A, B, C. Position Aaligns a hole of sufficient diameter with the tubing so as to allow thevarious components of the synthesis to pass unimpeded. Position B alignsa porous frit to which is covalently attached oligonucleotides ofdeoxyadenosine (dA) approximately 20 bases in length. The quantity ofoligo dA is sufficient to anneal the entire quantity of oligo dT,attached to the initiating substrate as described above. In position B,only solutes can pass through and no immobilization material (e.g. thosecontained in solutions 151, 153, 155, 157). Position C closes all flow.Reaction chamber 111 contains the initiating substrate in water,solution 161. As mentioned above the initiating substrate contains oligodT which is ≧20 nucleotides in length. Stock containers 121, 123, 125,127, 128 and 129 are connected to the reaction vessel 115 by way ofperistaltic tubing or some similar material to effect transport of thereagents contained in the stock containers. Additionally, vessel 115 isconnected to tubing, 181, which contains a distributor, 171 which servesto divert the flow of solutes either to tubing 183 or tubing 185 orrecycled back to stock containers after passing through adsorption media(e.g. hydroxylapatite) contained in 130, 132, 134 or 136 via tubes 187,189, 191 or 193. Tubing 183 feeds back to vessel 115; tubing 185 feedsinto a discard container. Solute movement through the tubing isfacilitated by pumps, 131, 133, 135, 137, 138, 139, 163 (e.g.peristaltic pumps) or similar devices which will force fluids, gels orviscous suspensions through tubing to desired destinations.

The automated process for synthesis involves the following flow ofsolutes and stopcock positions controlled by at least onemicroprocessor. The microprocessor controls pumps, the distributors andthe stopcock positions:

1) Stopcock 113, position C (blocked); stopcock 171 in discard position(tube 185); tethered nucleotide, buffers (solution 151, 153, 155, or157) are combined with substrate oligonucleotide or polynucleotide(solution 161) to yield a tethered oligonucleotide or polynucleotide.

2) Stopcock 113, position B (oligo-dA frit); distributor 171 in recycleposition (tube 187, 189, 191 or 193); unreacted polynucleotide isadsorbed in containers 130, 132, 134 or 136; enzyme, buffers arerecycled.

3) Stopcock 113, position B (oligo-dA frit); stopcock 171 in discardposition (tube 185); cleavage buffer (solution 158) added to immobilizedpolynucleotide yielding a free polynucleotide annealed to the oligo-dAfrit; released mononucleotides discarded.

4) Stopcock 113, position B (oligo-dA frit); stopcock 171 in recycleposition (tube 183); water (solution 159) is passed through the chamberand frit to release the annealed polynucleotide from the frit and returnthe polynucleotide to the reaction chamber. The free polynucleotideresides in tubing 183 during Step 5.

5) Stopcock 113, position A (completely open); stopcock 171 in discardposition (tube 185); immobilized substrate discarded prior to entry ofpolynucleotide back into reaction chamber.

6) The final product is recovered via tube 185 with stopcock 113 inposition A.

It will be appreciated that for these separation techniques to beeffective, the starting oligonucleotide or polynucleotide substrateshould consist of at least approximately 20 nucleotides. The compositionof the starting oligonucleotide or polynucleotide can be anything thatwill enable the subsequent purification steps as well as the ultimatecleavage of the object oligonucleotide or polynucleotide from thestarting oligo- or polynucleotide. An example of a nucleotidemodification that would enable final separation of startingoligonucleotide or polynucleotide from the object polynucleotide isbiotinylation of the primary amines of dA, dC, or dG. Additionally, astarting oligonucleotide substrate containing 7-methyl guanosine at the3' end will provide a cleavage site, as described previously, forultimate recovery of the object polynucleotide.

Thus, it can be appreciated that, regardless of the equipment employed,the method of the present invention efficiently produces oligonucleotideor polynucleotides in high yield, with a significant reduction in thenumber of unreacted sequences per cycle. This greatly simplifies theultimate isolation of the object nucleotide chain for furtherexperimentation. Once isolated, the nucleotide chain may be "stitched"together with other polynucleotides and formed into double stranded DNAas described above or may be amplified by conventional means such as bypolymerase chain reactions for use in recombinant DNA end useapplications.

E. Kits

The present invention also contemplates a kit for carrying out thepresent inventive procedure. Typically, a kit would contain all thesolutions and substances needed for performing the instant synthesisprocedure together with instructions for carrying out the procedure. Atypical kit for carrying out the claimed process would include aninitiating substrate of the present invention, various nucleoside 5'triphosphates of the present invention having a removable blockingmoiety protecting the 3' position, an enzyme of the present inventioncapable of catalyzing the formation of a 5' to 3' phosphodiester linkagebetween the unprotected 3' hydroxyl group of the initiating substrateand the 5' phosphate of the blocked 5'-triphosphate. Additionalcomponents and solutions optionally included in the kit are variousrequired reaction solutions and reaction buffers, reaction vessels inwhich to perform the assay, deblocking chemicals, solutions or enzymesof the present invention.

A kit for carrying out the instant synthesis may also contain initiatingsubstrates that are attached to a solid support. The kit may contain avariety of initiating substrates attached to solid supports, so that thefirst nucleoside of a desired oligonucleotide can be selected byselecting the appropriate initiating substrate.

In other kits for carrying out the present process, initiatingsubstrates having oligonucleotides of a preselected nucleotide sequenceare provided to allow oligonucleotides and polynucleotides having thispreselected nucleotide sequence incorporated into its 5' to be produced.Kits with this type of initiating substrate can provide easy synthesisof oligonucleotides having, for example, a restriction endonucleasecleavage site present in its nucleotide sequence.

Other kits contemplated by the present invention include initiatingsubstrates having various derivatized nucleotides, nucleoside analogs,or non-nucleoside molecules that allow oligonucleotides produced usingthose initiating substrates to have useful properties such as easilycoupling to other molecules, unique biologic activity or other uniquefeatures. Other kits would have an initiating substrate of the presentinvention such as double-stranded oligonucleotides.

The present invention also contemplates kits for producing nucleoside5'-phosphate and nucleoside analogs having a removable blocking moietyprotecting its 3' position. These kits would allow a user to producenucleoside 5' triphosphates and equivalents that are useful in thepracticing of the present invention.

The present invention also contemplates kits that contain additionalcomponents for carrying out other molecular biologic procedures inconjunction with the methods of the present invention. For example,components of the present invention may be present in a kit thatcontains vectors and concomitant cell lines for expression of a proteinor enzymes for desired modifications of amplification of the nascent orfully synthesized object polynucleotide.

The following examples further illustrate the present invention, and arenot to be construed as limiting the scope thereof. Unless otherwiseindicated, materials were obtained from Promega, Fisher, Aldrich, Sigma,Pharmacia, Gibco-BRL, Bio-Rad and New England Biolabs. All parts andpercentages are by weight unless expressly indicated to be otherwise,and all temperatures are in degrees Celsius.

EXAMPLES Example 1 Synthesis of Protected Nucleotides

A. Synthesis of Protected Nucleotides by Reaction of the 3' Hydroxylwith Carboxylic Acids

i. Toluic acid. One hundred μL of 1M toluic acid (either the para orortho isomer) in anhydrous N,N-dimethylformamide (DMF) was mixed, in anitrogen atmosphere, with 100 μL of 1M carbonyldiimidazole, also inanhydrous DMF. Formation of the imidazolide was allowed to proceed atroom temperature for 30 seconds. To this mixture was added 100 μL of a50 mM solution of deoxynucleoside 5'-triphosphate in water. Formation ofthe toluoyl-dNTP ester proceeded at room temperature for 12 hours.

The triphosphates (including both 3'-hydroxy unreacted triphosphates andthe 3'-toluoyl triphosphates) were separated from the other reactioncomponents by precipitation in the presence of 9 volumes of acetone. Theinsoluble nucleoside triphosphates were recovered by centrifugation andremoval of the soluble components. The nucleosides were then redissolvedin 100 μL of water, toluoyl ester was separated from the startingnucleotide by chromatography on Whatman 3MM cellulose paper which hadbeen prewashed first in isopropanol, butanol, and water in theproportion of 2:2:3 by volume, and then in water alone, prior to drying.The solvent to achieve separation by ascending chromatography containedisopropanol, butanol, and water also in the proportion of 2:2:3 byvolume. Detection of the various separated components was by ultravioletlight absorption at 254 nm. The dNTP-3'-O-toluate was cut from the paperand eluted into water. After concentration to dryness in vacuo, thenucleotide ester was redissolved in water to a final concentration of.sup.˜ 0.1-1 mM. This material was then subjected to mass spectroscopicanalysis to confirm the structure. The predicted mass numbers for thetoluoyl esters of DATP, dCTP, dGTP, and TTP are 608, 584, 624, and 599respectively. In each case these mass numbers were observed. These massnumbers were not observed in the spectra obtained from the unprotecteddeoxynucleoside triphosphates.

In related experiments, a variety of esters have been formed fromcarboxylic acids to yield aromatic or aliphatic protecting groups at the3' position.

ii. Benzoic acid and dimethylbenzoic acid. Benzoic acid as well as the2,6- and 3,5-dimethylbenzoic acid isomers were esterified to nucleotidetriphosphates by the same methods described above in order to evaluateposition effects of methyl groups on the overall kinetics of subsequentenzyme reactions.

iii. 4-Nitrobenzoic acid. Esterified to the 3'-hydroxyl using the samemethods.

iv. 2 -Napthoic acid. Esterified to the 3'-hydroxyl using the samemethods.

v. Isovaleric acid. Esterified to the 3'-hydroxyl using the samemethods.

Depending on the particular stability requirements, the procedures arereadily adaptable to the utilization of virtually any carboxylic acidfor esterification and protection of the 3'-hydroxyl of a nucleotidetriphosphate.

B. Synthesis of Protected Nucleotides by Reaction of the 3'-Hydroxylwith an Ether

2.5 mg of deoxynucleoside triphosphate was dissolved in 100 μL anhydrousDMSO containing 5.2 mg para-toluene sulfonic acid. The solution wascooled to 0° C.; 200 μL of ethyl vinyl ether was then added and allowedto react for 3 minutes. 200 μL of 1M Tris-Cl, pH 9.0 was then added withvigorous shaking resulting in the formation of two liquid phases. Theether phase was discarded and to the aqueous phase was added 10 volumesof absolute ethanol to precipitate the nucleotides. After incubation for10 minutes at -20° C. the nucleotide pellet was obtained bycentrifugation, and was redissolved in 0.25M NaCl followed by theaddition of 10 volumes of ethanol. The final precipitated nucleotidepellet was dissolved in 100 μL of water and applied to Whatman 3MM paperfor chromatography to separate the nucleotide ether from unreactednucleotides. Ascending chromatography was performed as described abovewith a solvent of isopropanol, butanol, and water in the proportion of2:2:3 by volume. The nucleotide ether, where the protecting group is anethoxyethyl moiety, migrates with an R_(f) (relative to the unreactednucleotide) of 1.25. This species was cut out of the paper and elutedwith water to yield the purified derivative.

C. Synthesis of Protected Nucleotides by Phosphorylation of the3'-Hydroxyl

i. Chemical synthesis. 2.0 mg of deoxynucleoside triphosphate wasdissolved in 60 μL anhydrous DMSO, 2 μL orthophosphoric acid, and 6 μLtriethylamine. To start the reaction, 6 μL of trichloroacetonitrile wasadded and the mixture was incubated at 37° C. for 30 minutes. Thereaction was cooled to room temperature and 5 μL of 5M NaCl was addedfollowed by 1.4 mL of acetone. The precipitation of nucleotide wasallowed to proceed at -20° C. for 10 minutes; nucleotide was recoveredby centrifugation, redissolved in 100 μL 0.25M NaCl and reprecipitatedby the addition of 1.4 mL of absolute ethanol. The final nucleotide wasrecovered by centrifugation, dissolved in 100 μL of water and applied toWhatman 3MM paper. Separation of nucleotide tetraphosphate(5'-triphosphate, 3'-monophosphate) was by ascending chromatography inisopropanol, butanol, and water in the proportion of 2:2:3 by volume.The nucleotide tetraphosphate migrates with an R_(f) (relative to theunreacted nucleotide) of 0.90.

Alternatively, the same phosphorylation reaction components (phosphoricacid, teithylamine, and deoxynucleotide) can be dissolved in formamideand the reaction allowed to proceed at 70° C.

Alternative chromatography solvents include 1-propanol, concentratedammonia, water (55:20:25), in which case the Rf of the tetraphosphate or3 mm paper is approximately 0.8 relative to unreacted triphosphate.

ii. Enzymatic synthesis. Deoxynucleoside 3'-monophosphates (Sigma) werephosphorylated at the 5' position using polynucleotide kinase. Thereaction was performed at pH 9.0 to minimize the inherent 3' phosphataseactivity of the enzyme, in a solution consisting of 50 mM Tris-Cl (pH9.0), 10 mM MgCl₂, 1.5 mM spermine, 5 mM dithiothreitol, 3 mM 3'-dNMP,30 mM ATP, and 20 units of polynucleotide kinase (Sigma or Pharmacia) ina final volume of 200 μL for 16 hours at room temperature. Thephosphorylation was monitored by chromatography (Whatman 3MM paper)after removal of the ATP by chromatography through Affi-Gel 601(Bio-Rad).

The nucleoside 5'-monophosphate 3'-monophosphate was furtherphosphorylated at the 5' position using nucleoside monophosphate kinaseand pyruvate kinase in a solution containing 50 mM Tris-Cl (pH 7.4), 10mM MgCl₂, 1.5 mM spermine, 5 mM dithiothreitol, 30 mM ATP, 4 mMphosphoenolpyruvate, 10 mM KCl 150 μg/mL pyruvate kinase (Sigma), and100 μg/mL nucleoside monophosphate kinase (Boehringer Mannheim). Thereaction proceeded at room temperature for 30 minutes (for dA) to 4hours (for dT, dC, dG). The deoxynucleotides were again separated fromATP by chromatography on Affi-Gel 601 followed by concentration todryness in vacuo and dissolution in 200 μL of water. Purification of thetetraphosphate from other nucleotides was by paper chromatography asdescribed above.

D. Synthesis of a Benzoylated Nucleotide Tethered to Agarose Beads

One hundred μL of 1M p-aminobenzoic acid in 10 mM sodium hydroxide, 90%DMF, pH 10, was mixed with 100 μL of 1M N-succinimidyl3-(2-pyridylthio)propionate also in basic DMF. Coupling of thesuccinimidyl to the amine was allowed to proceed at room temperature forfour hours. The reaction was monitored and the coupled product purifiedby thin layer chromatography on silica gel using a mixture of chloroformand methanol as the solvent. Silica gel containing the coupled productwas extracted with DMF, filtered, and added to an equal volume (.sup.˜100 μL) of 1M carbonyl diimidazole in anhydrous DMF followed immediatelyby the addition of 100 μL of 50 mM deoxynucleoside triphosphate. Theproduct, a dNTP coupled by an ester linkage to a tether containing anamide and a disulfide bond, was treated with 2-mercaptoethanol in anitrogen atmosphere to expose the sulfhydryl, and subsequently purifiedby purification from 10 volumes of absolute ethanol. The purifiedproduct, under nitrogen atmosphere, was then incubated with 0.2 mLAffi-Gel™ 501, an organomercurial crosslinked agarose, in 50 mM sodiumphosphate, pH 6, at room temperature for one hour to allow covalentmercaptide bonds to form.

E. Production of Nucleoside Having a Removable 3' Blocking Moiety UsingAlternate Strategies

An alternative strategy for making ester and ether triphosphatesinvolves chemical phosphorylation of a nucleoside which already containsan ester or ether protecting group at the 3' position.

Isovaleroyl Ester. One hundred μL of 1M isovaleric acid dissolved inanhydrous N,N-dimethylformamide (DMF) under a nitrogen atmosphere ismixed with 100 μL of 1M carbonyldiimidazole, also in anhydrous DMF.Formation of the imidazolide is allowed to proceed at room temperaturefor 30 seconds. To this mixture is added 100 μL of 5'-O-dimethoxytritylthymidine (DMT thymidine; Sigma Chemical Co.) also dissolved inanhydrous DMF. Formation of the isovaleroyl ester at the 3' hydroxylproceeds at room temperature for 12 hours.

The 5'-DMT 3'-isovalerate thymidine is purified by thin layerchromatography or HPLC and recovered from solvents in vacuo. To removethe 5' protecting group, the compound is reacted in 1 mL of methylenechloride with 5 equivalents of finely powdered, anhydrous zinc bromidewith stirring at room temperature. The reaction is monitored by TLC todetermine the optimum time for specific removal of the DMT group. The5'-OH 3'-isovaleroyl ester of thymidine is then recovered after TLC orHPLC purification. These procedures are generally applicable to all fournucleosides.

To phosphorylate the 5' hydroxyl, the ester is dissolved or resuspendedon 0.5 ml triethyl phosphate. 0.4 mmol phosphoryl chloride (POCl₃) isadded and the reaction is allowed to proceed at room temperature for1-14 hours. Purification of the 5'-monophosphate-3'O-isovaleroyl esteris by chromatography on DEAE Sephadex A-25 eluted with a linear gradientof triethylammonium carbonate (pH 7-8). The monophosphates are eluted ata buffer concentration of approximately 0.2-0.3M.

The purified monophosphate ester (0.1 mmol) is converted into itspyridinium salt with the pyridinium form of Dowex-50W X-8 cationexchange resin. The tributylammonium salt is prepared by addition oftributylamine (0.2 mmol), and the product is dried by addition andevaporation of anhydrous pyridine and N,N-dimethylformamide. To asolution of the anhydrous tributylammonium salt in N,N-dimethylformamide(1 ml) is added 1,1-carbonylbis(imidazole) (0.5 mmol). Formation of theimidazolide may be monitored by TLC chromatography or HPLC. After thereaction is complete, 35 μL of methanol is added to react with remainingcarbonylbis(imidazole) and the solution is kept at room temperature for5 minutes. Tributylammonium pyrophosphate (0.5 mmol) inN,N-dimethylformamide (5 ml) is then added dropwise with stirring. Themixture is kept for several hours at room temperature then evaporated todryness. The triphosphate is isolated by chromatography as above and iseluted at approximately 0.4-0.5M triethylammonium carbonate buffer.These phosphorylation procedures are generally applicable to all fournucleosides.

Depending on the particular stability requirements, the proceduresdescribed above are readily adaptable to the utilization of virtuallyany carboxylic acid for esterification and protection of the 3'-hydroxylfollowed by the chemical phosphorylation of the 5' hydroxyl to producethe 3' protected nucleotide triphosphate. These carboxylic acids includebut are not limited to the following classes: formic, benzoylformic,chloroacetic, fluoroacetic, methoxyacetic, phenoxyacetic,chlorophenoxyacetic, phenylacetic, propionic, butyric, isovaleric,succinic, pentanoic, pivalic, adamantane carboxylic, crotonic, butenoic,substituted benzoic (e.g. nitrobenzoic, methylbenzoic, chlorobenzoic,phenylbenzoic), napthoic acids.

As is understood by those of ordinary skill in the art, there arenumerous methods for creating esters for the purpose of transientlyprotecting hydroxyl groups. In addition, there are numerous strategiesfor transiently protecting one hydroxyl group (e.g. the 5' hydroxyl of anucleoside) in order to introduce an ester at another hydroxyl group(e.g. the 3' hydroxyl of a nucleoside).

Silyl Ether. 1 mmol of thymidine is dissolved in 1 ml ofN,N-dimethylformamide at room temperature. To this solution is added 20mmol of imidazole and 10 mmol of tert-butyldimethylsilylchloride(t-BDMS). The reaction is allowed to proceed at room temperature untilboth hydroxyl groups are silylated.

The disilylated nucleoside is recover by TLC or HPLC and the solventsremoved in vacuo. The 5' silyl protecting group is then selectivelyremoved by incubation in 80% acetic acid. The reaction is monitored byTLC to determine the optimum time for removal of the 5' protectinggroup. The resulting 3' silylated nucleoside is purified by TLC or HPLC,phosphorylation at the 5' position is by the procedures described aboveyielding a nucleotide triphosphate 3'-t-BDMS ether. These procedures aregenerally applicable to all four nucleosides.

Methoxymethyl Ether. 1 mmol of DMT-thymidine is dissolved indiisopropylethylamine and reacted with 4 mmol chloromethyl methyl ether(Aldrich Chemical Co.) at 0° C. for 1 hour followed by warming to roomtemperature and further reaction for 8 hours. The thymidine 5'-DMT3'-methoxymethyl ether is purified by TLC or HPLC and recovered fromsolvents in vacuo.

To remove the 5' protecting group, the compound is reacted in 1 mL ofmethylene chloride with 5 equivalents of finely powdered, anhydrous zincbromide with stirring at room temperature. The reaction is monitored byTLC to determine the optimum time for specific removal of the DMT group.The 5'-OH 3'-methoxymethyl ether of thymidine is then recovered afterTLC or HPLC purification. Phosphorylation at the 5' position isaccomplished by the procedures described above yielding a nucleotidetriphosphate 3'-methoxymethyl ether. These procedures are generallyapplicable to all four nucleosides.

As is understood by those of ordinary skill in the art, there arenumerous methods for creating ethers for the purpose of transientlyprotecting hydroxyl groups. In addition, there are numerous strategiesfor transiently protecting one hydroxyl group (e.g. the 5' hydroxyl of anucleoside) in order to introduce an ether at another hydroxyl group(e.g. the 3' hydroxyl of a nucleoside).

Ethers useful for protection of nucleoside 3' hydroxyl groups includebut are not limited to the following classes: methyl, substituted methyl(e.g. benzyloxymethyl, methoxyethoxymethyl, trimethylsilylethoxymethyl), pyranyl, furanyl, sustituted ethyl (e.g. ethoxyethyl,methoxyethyl, methylmethoxy ethyl), butyl, allyl, cinnamyl, benzyl andsubstituted benzyl (e.g. methoxybenzyl, halobenzyl, nitrobenzyl),anthryl and silyl ethers.

Example 2 Regeneration of the 3' Hydroxyl from Protected Nucleotides

A. Deprotection of nucleotide 3'-O esters. The ester protecting groupswere removed from the 3'-hydroxyl of dNTPs by incubation in 1 mM CoCl₂and 100 mM potassium cacodylate, pH 6.8. The conversion of the ester tothe hydroxyl was evaluated by cellulose and paper chromatography and wasfound to be nearly quantitative after 15 minutes of incubation at 37° C.These deprotection conditions were equally effective for each of thefour nucleotides. Further evaluation revealed that the instability wasdue to both the buffer and the divalent cation.

The instability of the toluoyl esters in other commonly used TdTasecoupling reaction buffers was explored. The relative degree ofinstability due to the various buffers (in the presence of 1 mM CoCl₂)was found to be cacodylate>tris(hydroxymethyl) aminomethane>sodiumacetate or phosphate. Instability due to the cations (in cacodylatebuffer) was found to be Co⁺⁺ >Mn⁺⁺ >Mg⁺⁺. Degradation of the esters wasfirst observable after about three minutes of incubation. Incubation inbuffer alone or cation alone produced no observable degradation. Incommon with many other types of esters, these deoxynucleotide esterswere also sensitive to basic conditions e.g. incubation in 10-100 mMNaOH. The other esters of dNTPs (isovaleroyl, dimethylbenzoyl, napthoyl,and nitrobenzoyl) were also relatively unstable in the buffered divalentcations.

In general, these results identify unexpected properties of the estersof dNTPs and provide a convenient and gentle method for the rapidremoval of these esters from the growing polynucleotide chain after acoupling reaction. These deprotection conditions are sufficiently gentleto enable the synthesis of an object polynucleotide onto a pre-existingdouble stranded DNA without denaturation of the DNA at every cycle. Thiscapacity for "add-on" synthesis using a double-stranded polynucleotideas the initiating substrate is demonstrated in Example 5 below.

B. Deprotection of nucleotide 3'-O ethers. The 3' ethoxy ethyl ether ofthe nucleoside triphosphates were stable in cobalt containing buffersbut could be readily removed by incubation in 5% acetic acid at roomtemperature or by incubation in 0.5N HCl/THF at 0° C.

C. Deprotection of nucleotide 3'-phosphates. The 3'-phosphate wasspecifically removed by incubation of the nucleotide in a solutioncontaining 50 mM sodium acetate, pH 5.5, 10 mM MgCl₂, and 20 units ofnuclease P1, an enzyme which specifically removes phosphates from the 3'position of mononucleotides. The reaction was allowed to proceed for 90minutes at 37° C. This enzyme would not be appropriate in the case of aprotected nucleotide attached to an initiating substrate since it isalso a phosphodiesterase. In this case an alternative phosphatase can beused, which is described below.

Example 3 Efficiency of Enzyme Catalyzed Phosphodiester Bond FormationUsing Protected Deoxynucleotidyl Triphosphates

The ability of a polymerizing enzyme, TdTase, to catalyze the creationof a phosphodiester bond between an initiating polynucleotide substrateand a 3'-O-protected deoxynucleotidyl triphosphate was measured using atransferase/ligase assay. In this assay, transfer of a nucleotide to aninitiating substrate DNA, such as a linearized vector, will inhibit theability of the vector to be relegated into a circular form. The relativequantity of circular vector DNA in each reaction can then be measured bybacterial transformation.

100 μM deoxynucleotide 3' ethoxy ethyl ether, or 3' phosphate wereincubated with Pst 1-digested Puc 8 vector DNA (1 μg) in the presence of1 mM CoCl₂, 0.1 mM DTT, potassium cacodylate, pH 6.8, and 40 unitsTdTase (Promega) in a total volume of 25 μL. In the case of the 3'ester, the same reaction was performed with the exception that the CoClswas replaced with MnCl2 and the cacodylate buffer was replaced withTris-Cl. The reactions were allowed to proceed at 37° C. for 15 minutesat which time they were terminated by the addition of 1 μL of 100 mM Na₂EDTA, 0.1% sodium dodecyl sulfate. The Puc 8 DNA was separated from theother components of the reaction by chromatography through aqueouspacked Sepharose™ CL-6B and was then used in a ligase reaction. Theligation reaction consisted of the Puc 8 DNA, 1 mM Na₂ ATP, 50 mMTris-Cl, pH 8.0, 1 mM MgCl₂, 100 μg/mL bovine serum albumin and 100units of T4 DNA ligase (New England Biolabs). The ligation reaction wasallowed to proceed at 16° C. for 18 hours. The Puc 8 DNA was againrecovered by Sepharose™ CL6B chromatography.

The inhibition of the ligation reaction due to the addition of anucleotide to the Puc 8 DNA by TdTase was quantified by a bacterialtransformation assay. Competent E. coli JM109 bacteria (Promega) wereincubated with 100 ng of the Puc 8 DNA according to the instructionsprovided with the transformation competent cells. Briefly, this involveda heat shock of the admixture for one minute at 42° C., incubation ofthe bacteria in LB broth at 37° C. for one hour, and overnight growth ofthe bacteria on LB agar Petri plates containing 50 μg/mL ampicillin.Colonies from each transformation were then counted.

    ______________________________________                                        dNTP in TdTase                                                                             Vector      Religation                                                                             Trans-                                      Reaction     Substrate   formed   Colonies                                    ______________________________________                                        none         Pst 1 - Puc 8                                                                             yes      1,381                                       (positive control)                                                            none         Pst 1 - Puc 8                                                                             no       325                                         (background)                                                                  dideoxy-ATP  Pst 1 - Puc 8                                                                             yes      342                                         dATP 3'-O toluate                                                                          Pst 1 - Puc 8                                                                             yes      316                                         dATP 3'-O ether                                                                            Pst 1 - Puc 8                                                                             yes      330                                         dATP 3'-phosphate                                                                          Pst 1 - Puc 8                                                                             yes      340                                         dATP-3'OH    Pst 1 - Puc 8                                                                             yes      636                                         ______________________________________                                    

The results demonstrate that the protected nucleotides are utilized byTdTase for the creation of phosphodiester bonds. The covalent attachmentof the protected nucleotide to the vector DNA blocks the vector fromreligation. In the case of the unprotected nucleotide (DATP 3'-OH) theenzyme may be predominantly adding homopolymer tails to a population ofvector molecules leaving some vectors unmodified.

The efficiency of the TdTase catalyzed transfer, as measured by thenumbers of colonies in excess of the background value, were comparablewhen comparing the protected mononucleotide with dideoxynucleotide. Theabsence of transformed colonies above the background value compared to acontrol which produced greater than 1000 colonies, indicates a TdTasecatalyzed transfer of protected mononucleotide to ≧99.9% of theinitiating substrate 3' hydroxyls.

Example 4 Inhibition of Phosphodiester Bond Formation by ProtectedNucleotides

Attachment of a protected mononucleotide to vector DNA will prevent thesubsequent attachment of a biotin labelled nucleotide so long as theprotecting group is affixed to the 3'-hydroxyl. This inhibition ofvector biotinylation can be readily quantified by blotting assays afteragarose gel electrophoresis.

Vector DNA (either Puc 8 or pBluescript) digested with the appropriaterestriction enzyme, was reacted with approximately 100 μM protectednucleotide for varying times in the presence of 25 units TdTase (Promegaor BRL) in appropriate buffers in a final volume of 25 μL. To thereaction mix was then added 1 μL of 300 μM biotinylated dUTP (Sigma orBoehringer) and the reaction was allowed to proceed for 1-3 minutes atwhich point the reaction was stopped by the addition of 1 μL of 1%sodium dodecylsulfate, 50 mM Na₂ EDTA. The mixture was heated to 75° C.for 1 minute then electrophoresed in an agarose gel to visualize theDNA. In related assays, the vector DNA was purified from the othercomponents of the reaction prior to the addition of biotinylatednucleotide. Purification was by centrifugal chromatography on SepharoseCL-6B. This step was included to avoid the possibility that lowmolecular weight inhibitors were slowing the activity of the TdTase.

The incorporation of biotin into the DNA was measured using a standarddye reaction procedure. The DNA was first blotted onto a piece ofnitrocellulose paper. The nitrocellulose paper with the DNA adhering toit was then heated to 80° C. in a drying oven for 30 minutes andre-wetted in 25 mL of 50 mM Tris-Cl pH 8.1, 150 mM NaCl, 0.1%Triton-X-100 (TBST) and 10% (w/v) Carnation non-fat dry milk, a solutionwhich is intended to enhance the contrast of the final dye reaction.After 1 hour of incubation in the milk solution, a fresh solution ofTBST containing approximately 1 μg/mL of streptavidin alkalinephosphatase (Fisher Scientific #OB5000-ALPH) was added to the paper.Binding of streptavidin to biotin proceeded for 1 hour at roomtemperature. The paper was then transferred to 25 mL of fresh TBST for10 minutes to wash off excess streptavidin-alkalin phosphatase. Thiswashing step was repeated four times. The paper was then transferred to10 mL of 100 mM Tris-Cl, pH 9.5, 150 mM NaCl, 5 mM MgCl2, 300 μg/mLnitrotetrazolium blue and 150 μg/mL bromochloroindolyl phosphate tovisualize the quantity of bound streptavidin phosphatase by enzymaticrelease of the chromophoric bromochloro indole.

The results of the inhibition assays using a variety of blocking groupsis summarized below.

    ______________________________________                                        3' protecting                                                                             Biotinylation                                                                             Reaction                                              group (%)   time (min)  time (min)                                                                             Inhibition                                   ______________________________________                                        para-toluoyl                                                                              0.5-5       0.5-5    >50%                                         benzoyl     0.5-5       0.5-5    >50%                                         isovaleroyl 0.5-5       0.5-5    >50%                                         dimethylbenzoyl                                                                           0.5-5       0.5-5    >50%                                         ethoxyethyl 0.5-5       0.5-5    >50%                                         phosphate   0.5-5       0.5-5    >50%                                         ______________________________________                                    

Example 5 DNA Synthesis Using Protected dNTPs: Synthesis of a NewRestriction Site in the Puc 8 Vector

To demonstrate the synthesis of a desired DNA sequence directly onto avector DNA by the TdTase catalyzed addition of the protected dNTPs, weperformed sequential reactions on Pst 1-digested Puc 8 DNA in order tointroduce a new restriction site into the vector. The sequence at thetermini of the Pst1 Puc 8 DNA is: ##STR15## where the dotted linesindicate the annealed complementary strands of the vector. Sequentialcoupling and cleavage reaction were performed using the toluoyl estersof dNTPs as follows:

First coupling reaction--100 mM potassium cacodylate, pH 6.8, 1 mMCoCl₂, 0.1 mM DTT, 0.1 mg/mL BSA, 100 μM dTTP-3'O-toluate, 40 unitsTdTase (Promega), 37° C., 2 minutes.

Stop reaction--1 μL 100 mM Na₂ EDTA, 1 μL 10% sodium dodecyl sulfate,65° C., 2 minutes.

DNA recovery--Centrifugation through 0.5 mL packed Sepharose™ CL-6B inwater.

Ester cleavage reaction--100 mM potassium cacodylate, pH 6.8, 1 mMCoCl₂, 0.1 mM DTT, 0.1 mg/mL BSA, 37° C., 30 minutes.

Second coupling reaction--100 μM dGTP-3'O-toluate, 40 units TdTase(Promega), 37° C., 2 minutes.

Repeat stop, recovery and cleavage.

Third coupling reaction--100 μM dCTP-3'O-toluate, 40 units TdTase(Promega), 37° C., 2 minutes.

Repeat stop, recovery and cleavage.

Fourth coupling reaction--100 μM dATP-3'O-toluate, 40 units TdTase(Promega), 37° C., 2 minutes.

Repeat stop, recovery and cleavage.

Final recovery of DNA--Centrifugation through 0.5 mL packed Sepharose™CL-6B in water.

A similar series of reaction were performed using the 3'-phosphates ofthe dNTPs with some modifications.

First coupling reaction--100 mM potassium cacodylate, pH 6.8, 1 mMCoCl₂, 0.1 mM DTT, 0.1 mg/mL BSA, 100 μM dTTP-3'-phosphate, 40 unitsTdTase (Promega), 37° C., 2 minutes.

Stop reaction--1 μL 100 mM Na₂ EDTA, 1 μL 10% sodium dodecyl sulfate,65° C., 2 minutes.

DNA recovery--Centrifugation through 0.5 mL packed Sepharose™ CL-6B inwater.

Phosphate cleavage reaction--0.1 m Tris.Hcl, pH 9.0, 0.1 m Nacl, 10 mMMgCl₂, and 20 units of alkaline phosphatase, 37° C., 30 minutes.

Second coupling reaction--100 μM dGTP-3'-phosphate, 40 units TdTase(Promega), 37° C., 2 minutes.

Repeat stop, recovery and cleavage.

Third coupling reaction--100 μM dCTP-3'-phosphate, 40 units TdTase(Promega), 37° C., 2 minutes.

Repeat stop, recovery and cleavage.

Fourth coupling reaction--100 μM dATP-3'phosphate, 40 units TdTase(Promega), 37° C., 2 minutes.

Repeat stop, recovery and cleavage.

Final recovery of DNA--Centrifugation through 0.5 mL packed Sepharose™CL-6B in water.

The modified vector DNA was intended to have the following new DNAsequence: ##STR16##

To demonstrate the presence of this new sequence in the vector, themodified Pst 1-Puc 8 was religated as previously described for 18 hoursat 16° C. The resulting recircularized or concatemerized plasmid wouldhave the following new structure in the Puc 8 polylinker: ##STR17##where the underlined portion is the recognition sequence for the Sph 1restriction enzyme, which did not previously exist in the vector.

The relegated vector was passed through a CL-6B spun column andincubated in the Sph 1 restriction enzyme buffer and 10 units of Sph 1(New England Biolabs). Agarose gel electrophoresis revealed that theoriginal Puc 8 DNA contained no Sph 1 recognition sequences and that therecovered DNA after the TdTase reactions contained the desired sequence.

To demonstrate the significance of the blocking groups, an identicalprotocol was followed using unblocked nucleotide triphosphates in thesynthesis reactions. The final religation product contained nodetectible Sph 1 sequences.

The foregoing examples and description of the preferred embodimentshould be taken as illustrating, rather than as limiting, the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. All such modifications are intended to be included withinthe scope of the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 8                                                  (2) INFORMATION FOR SEQ ID NO: 1:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (D) OTHER INFORMATION: base number 12 is m7g                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      CCCCCCCCCCCG12                                                                (2) INFORMATION FOR SEQ ID NO: 2:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      CCCCCCCCCCCCCCCTGCA19                                                         (2) INFORMATION FOR SEQ ID NO: 3:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CTGCAGGGGGGGGGGGGGGG20                                                        (2) INFORMATION FOR SEQ ID NO: 4:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      CCCCCCCCC9                                                                    (2) INFORMATION FOR SEQ ID NO: 5:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 5:                                      GGGGGGGGG9                                                                    (2) INFORMATION FOR SEQ ID NO: 6:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5                                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 6:                                      CTGCA5                                                                        (2) INFORMATION FOR SEQ ID NO: 7:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9                                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 7:                                      CTGCATGCA9                                                                    (2) INFORMATION FOR SEQ ID NO: 8:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10                                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) SEQUENCE DESCRIPTION: SEQ ID NO: 8:                                      CTGCATGCAG10                                                                  __________________________________________________________________________

We claim:
 1. A mononucleoside 5'-triphosphate having a removableblocking moiety protecting the 3' position which is an ester and whichhas the following formula: ##STR18## wherein R₂ is a triphosphate and Ris selected from the group consisting of: formate, benzoylformate,chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate,methoxyacetate, triphenylmethoxyacetate, phenoxyacetate,p-chlorophenoxyacetate, 2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,p-P-phenylacetate, 3-phenylpropionate, 3-benzoylpropionate, isobutyrate,4-oxopentanoate, pivaloate, adamanioate, crotonate, 4-methoxycrotonate,(E)-2-methyl-2-butenoate, o-(dibromomethyl)benzoate,o-(methoxycarbonyl)benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoateand α-naphthanoate.
 2. A mononucleoside 5' triphosphate having aremovable blocking moiety protecting the 3' position which is an esterand which has the following formula: ##STR19## wherein R₂ istriphosphate; and wherein R is selected from the group consisting of: H,CH₃, CH₃ (CH₂) where N is an integer from 1 to 12, (CH₃)_(x+1) (CH)_(x)where x is an integer from 1 to 12, (CH₃)_(x+1) (CH)_(x) (CH₂)_(n) wherex and n are independent integers from 1 to 12, C_(x) (CH₃)_(3x-)(x)1)(CH₂)_(n) where x and n are independent integers from 1 to 12, and##STR20## where R1, R3, R4, R5 and R6 are selected from the groupconsisting of CH₃, H or NO₂.
 3. The mononucleoside 5'-triphosophate ofclaim 1 or 2 wherein R₂ is a triphosphate and said mononucleoside5'-triphosphate is a deoxynucleoside.
 4. A mononucleoside5'-triphosphate having a removable blocking moiety protecting the 3'position which has the following formula: ##STR21## wherein R₂ istriphosphate; and wherein R1 is toluic, benzoic or acetic acid ester. 5.The mononucleoside 5'-triphosphate of claim 1, 2 or 4 wherein saidremovable blocking moiety is removable by an enzyme.
 6. Themononucleoside 5'-triphosphate of claim 1, 2 or 4 wherein said removableblocking moiety is removable by a reaction which occurs within 2 to 10minutes.
 7. The mononucleoside 5'-triphosphate of claim 1, 2 or 4wherein said removable blocking moiety is linked to a solid support.